CETSA® beyond Soluble Targets: a Broad application

to Multi-pass Transmembrane proteins

Aarti Kawatkar1*, Michelle Schefter1, Nils-Olov Hermansson2, Arjan Snijder2, Niek Dekker2, Dean G.
Brown1, Thomas Lundbäck2, Andrew X. Zhang1*, M. Paola Castaldi1*

1 Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Boston, USA

2 Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden

Supplemental Information

Supplemental information includes detailed materials and methods; supplementary figures S1−S8, tables
S1, S2.

Supplemental Information

Supplemental materials and methods

Table S1: Sources of loading control antibodies
Antibody Supplier Product Code Species Monoclonal/Polyclonal
Primary, CST (COX IV) 5247S Rabbit Polyclonal
Primary, CST (β Tubulin) 5346S Rabbit Polyclonal
Primary, CST (GAPDH) 8884S Rabbit Polyclonal
Primary, CST (β Actin) 12620 Rabbit Polyclonal
Primary, CST (Histone H3) 4499S Rabbit Polyclonal
Primary, abcam (SOD) Ab13498 Rabbit Polyclonal
Primary, abcam (Vinculin) Ab76320 Rabbit Polyclonal
Secondary, CST 7074S Anti-Rabbit Polyclonal

tFSEC on PAR2
Human PAR2 with five stabilizing mutations (V53A, G89A, H108A, M166L and V176E) and an N-terminal FLAG tag
and a C-terminal His10 sequence was transiently transfected in HEK293 6E cells. Cells were harvested after 48 hrs of
culturing. Cells were resuspended in 40 mM HEPES pH 7.4, 300 mM NaCl, 20% (v/v) glycerol, 1 tablet protease
inhibitor and benzonase and lysed using an ultrathurrax. After removal of cell debris, membranes were collected by
ultracentrifugation for 30 min in Ti45 Beckman rotor at 44k rpm at 4°C. The membranes were resuspended in 20
mM HEPES pH 7.4, 300 mM NaCl, 20% (v/v) glycerol + protease inhibitor and flash frozen in liquid nitrogen and
stored at -80°C.
Membranes were thawed and pre-incubated with either DMSO or 300 µM compound for 45 min on ice. 120 µl
solubilization buffer was added to 40 µl membrane compound mixture (solubilization buffer: 40 mM HEPES pH 7.4,
300 mM NaCl, 20% (v/v) glycerol, 1 tablet protease inhibitor/50 ml and 1.11% (w/v) DDM, 0.666% (w/v) CHAPS,
0.1333% (w/v) CHS), and the membranes were solubilized at 4°C for 30 min. The soluble fractions were collected by
centrifugation for 20 min at 50 krpm in TLA55 rotor at 4°C. The solubilized receptor was incubated at different
temperatures for 6 min, after which the samples are incubated with 0.9 µM FSEC probe for 1 hr after which 5-20 µl
was separated on an Agilent 1100 system equipped with an Agilent BioSEC 3 300Å 50x7.8 mm at 1.2 ml/min in buffer
containing 20 mm HEPES pH 7.4, 300 mM NaCl and 0.05% (w/v) DDM, 0.025% (w/v) DTM, 0.015% (w/v) CHAPS and
0.003%(w/v) CHS (Backmark, A. E., et al. (2013). "Fluorescent probe for high-throughput screening of membrane
protein expression." Protein Sci 22(8): 1124-1132.). Fluorescence with excitation 482 nm and emission 520 nm was
recorded, and the signal at elution time 1.3 min corresponding to PAR2 integrated. The integrated fluorescence
intensity was plotted as function of the incubation temperature and a melting temperature was estimated by a 6-
parameter fit in prism GraphPad.

Loading control experiment protocol

JEKO1 cells were cultured, harvested and collected (10 million per mL). Equal volume of cell suspension (60µL)
aliquoted into 0.2mL 12 PCR tubes. Samples were heated in PCR thermocycler for 3 mins to the reported
temperatures (~37 oC- 90 oC). The cells were lysed by freeze-thaw method and centrifuged to get supernatant
solutions, which were analyzed by Western blot.

TSPO CETSA: Thermal melt curve

HEK293 cells were seeded in four 10 cm dishes (Each 7 million cells mL-1 in PBS) one day before experiment with
fresh medium (DMEM + 10% FBS + 1% Glutamax + 1% P/S). On the day of the experiment, the medium of four
confluent 10 cm dishes was exchanged for 10 mL of fresh medium and 50 µL of vehicle (DMSO) or 50 L of 10 mM
compound was added to each of two dishes. Cells were incubated for 60 min at 37 oC and 5% CO2. Subsequently,
cells were washed with 10 cm PBS before addition of 2 mL of accutase solution to each dish. After 3 min of incubation
at 37 oC, 7 mL of medium with FBS was added to each dish, and cells were transferred into falcon tube and collected
by centrifugation at 340 g and 25 oC for 5 mins. After resuspension in 10 cm of PBS, two vehicle samples were
combined in one falcon tube, and the three inhibitor samples were combined in another. Cells were harvested by
centrifugation and washed once more with 10 ml PBS. After sedimentation, cells were resuspended in 300 L of PBS
supplemented with protease inhibitors. 12 times 20 L of each cell suspension was transferred into 0.2 mL PCR
tubes. One each of the inhibitor and vehicle samples was heated in PCR machine for 3 min to the reported
temperatures (37 oC- 75 oC) and then chilled at RT for 1 min. 5 L of ice-cold PBS supplemented with 1% (v/v) NP40
was added to each tube. Subsequently, samples were subjected to three freeze thaw cycles (snap frozen in liquid
nitrogen for ~ 3 min, briefly thawed in cold water bath at 25 oC). The entire content was centrifuged at 20, 000 g for
20 mins at 4 oC. After centrifugation, ~ 25 L of each supernatant soluble fraction was transferred into a new tube
and were subjected to gel electrophoresis. Supernatant was transferred to gel loading buffer (LDS) and protein
amounts were analyzed by SDS-PAGE followed by western blot. We used CST # 70358S Rabbit polyclonal (1:1000)
for detection of TSPO, Santa Cruz Antibody sc5573 (1:3000) for the detection of vinculin as a control, Ab13498
(1:1000) for the detection of SOD as another control, secondary antibody used was antirabbit from CST 7074
(1:5000). Western blots were performed on an iBlot2 device from Life technologies onto nitrocellulose membrane.
The membranes were incubated at 4 degrees overnight. Blocking and dilution of antibodies was performed in non-
fat milk.
The western blot intensities were obtained by quantifying the chemiluminescence count per square mm (I=
count per mm2) using ImageJ-win 64 (Fiji) and normalized to vinculin loading control. Compound data is represented
relative to TSPO intensity of the corresponding highest compound concentration i.e. 100 M, at either 37°C for initial
melt curves or 53 °C for dose response curves. The normalised data was plotted using Excel or GraphPad Prism.
Concentration response curves were fitted using a four-parameter non-linear regression curve fitting in GraphPad
Prism.

SERCA CETSA
A confluent plate of HeLa cells was washed, trypsinized, and taken up with growth medium (DMEM + 10% FBS + 1%
P/S). This suspension was spun down, washed with PBS, and taken up to 50 million cells mL-1 with DMEM + 1% P/S,
no FBS. This suspension was aliquoted into a series of PCR tubes (50 L) and to each suspension was added an equal
volume of 20 M Thapsigargin or 20 M CDN1163, or 0.2% (v/v) DMSO to make a 10 M final concentration of
Thapsigargin or CDN1163 (and 0.1% v/v final concentration of DMSO). The resulting suspensions were incubated at
37 oC, 5% CO2 for 1 hour, after which they were subjected to a 3 minute heat shock followed by rapid cooling to 25
oC. NP40 was then added to the suspensions to give a 0.25% (v/v) final concentration, and the suspensions were
mixed and subjected to three rapid freeze-thaw cycles in liquid nitrogen. The suspensions were then spun down at
11800 rcf and the supernatants were harvested for SDS-PAGE followed by Western Blot analysis. After the
completion of SDS-PAGE, gels were transferred to nitrocellulose membrane using the iBlot apparatus (20V for 3
minutes, 23V for 2 minutes, 25V for 2 minutes). Membranes were then blocked for 2 hours at room temperature
with 5% milk, followed by overnight incubation with primary antibody (rabbit anti-SERCA2 ATPase antibody, Abcam,
ab150435, 1:5000) at 4 oC. The primary antibody was removed, and the membrane was washed 3 times with Tris
Buffered Saline w/ 0.2% (v/v) Tween (TBST). The membrane was then incubated with secondary antibody (anti-
Rabbit HRP conjugated, CST 7074, 1:2000) for 1 hour at room temperature and washed 3 times with TBST. The
membrane was exposed with Western Blot Substrate for 3 minutes and luminescence signal was read.
The western blot intensities were obtained by quantifying the chemiluminescence count per square mm (I= count
per mm2) using ImageJ-win 64 (Fiji) and normalised to loading control. Signals were also normalised to the lowest
temperature to accurately assess the behavior. The normalised data was plotted using Excel or GraphPad Prism.
Concentration response curves were fitted using four parameter non-linear regression curve fitting in
GraphPad Prism.

PAR2 CETSA: Thermal curve

132N1 cells transiently transfected with plasmid encoding human wild type PAR2 were seeded in two 15 cm dishes
(Each 7 million cells mL-1 in PBS) one day before experiment with fresh medium (DMEM + 10% FBS + 1% Glutamax
+ 1% Penicillin and streptomycin solution). On day of experiment, the medium of confluent dishes was exchanged
for 15 mL of fresh medium containing 50 µM of compound or DMSO. Cells were incubated for 90 min at 37 oC and
5% CO2. Subsequently, cells were washed with 10 ml PBS, harvested with accutase solution (2 mL) at 37 oC,
quenched with medium with FBS (7 mL), and cells were transferred into falcon tube and collected by
centrifugation at 340 g and 25 oC for 5 mins, washed with PBS (2x 5 mL). The cells were resuspended in 400 uL of
PBS supplemented with protease inhibitors. 12 times 30 µL of each cell suspension was transferred into 0.2 mL
PCR tubes. One each of the inhibitor and vehicle samples was heated in PCR machine for 3 min to the reported
temperatures (37 oC- 85 oC) and then chilled at RT for 1 min. 7 µL of ice-cold PBS supplemented with 1% (v/v)
NP 40 was added to each tube. Subsequently, samples were subjected to three freeze thaw cycles (snap frozen in
liquid nitrogen for ~ 3 min, briefly thawed in cold water bath at 25 oC). The entire content was centrifuged at
20,000 g for 20 mins at 4 oC. After centrifugation, ~ 25 µL of each supernatant soluble fraction was transferred
into a new tube. The protein content was measured by Bradford test of supernatant of 37 oC sample. Soluble
fractions were subjected to deglycosylation using PNGase F (NEB P0704s). https://www.neb.com/products/
p0704-pngase-f#Product%20Information. Amount of protocol was adjusted depending upon the protein content.
3 µL of soluble fraction (12 µg of glycoprotein), 1 µl of Glycoprotein Denaturing Buffer (10X) and H2O (5 µl) were
combined to make a 10 µl total reaction volume. The glycoprotein was denatured at 100°C for 10 minutes which
was then chilled on ice and then centrifuged for 10 seconds, after which 2 µl GlycoBuffer 2 (10X), 2 µl 10%
NP-40 and 6 µl H2O were added. 1 µl PNGase F was then added, and the sample was mixed gently and
incubated at 37°C for 1 hour, after which the samples were then subjected to gel electrophoresis. Supernatant
was transferred to gel loading buffer (LDS) and protein amounts were analyzed by SDS-PAGE followed by western
blot. We used CST # D61D5 Rabbit monoclonal (1:1000) for detection of PAR2, SantaCruz antibody sc5573
(1:3000) for the detection of vinculin, secondary antibody used was antirabbit from CST 7074 (1:5000). Western
blots and data analysis were performed as described in general section.
PAR2 CETSA:
The cells were plated in 6 well plates and dilution series of 2-fold/ 3-fold dilutions were used, starting at 100 µM
final concentration. Cells were incubated for 90 min at 37 oC and 5% CO2. Subsequently, cells were washed with 10
ml PBS, harvested with accutase solution (2 mL) at 37 oC, quenched with medium with FBS (7 mL), and cells were
transferred into falcon tube and collected by centrifugation at 340 g and 25 oC for 5 mins, washed with PBS (2x 5
mL). Subsequent handling was identical to the procedures in obtaining the curves.

PAR2 mechanistic study:

132N1hPAR2 transiently transfected cells were cultured, harvested using accutase and collected as described
previously. Equal volume of cell suspension (100 µL) aliquoted into 0.2mL 24 PCR tubes. Samples were heated in
PCR thermocycler for 3 mins to the reported temperatures (~37 oC- 75 oC). In the last 30 seconds of heating, 1.6 µL
of 10 mM solution of AZ’8838 (or DMSO) was added to the PCR tubes (50 µM) and left at room temperature
for 10 minutes. The cells were lysed as described before by adding 1% (v/v) NP40 to make final concentration of
detergent as 0.2% (v/v). The cells were freeze-thaw as mentioned previously and centrifuged to get supernatant
solution, which was analyzed by Western blot.

Supplemental Figures
 Figure S1. Optimisation of TSPO CETSA Western Blot conditions

(A & B) In-cell CETSA with detergents DDM and NP-40, (C & D) In-lysate CETSA with detergents DDM and NP40. (E)
Isothermal Dose Response of TSPO to Alpidem treatment at 70 oC.
Figure S2. In addition to popular loading controls Actin and Vinculin, SOD1, COX IV, Histone could be used as an
alternative loading control protein. SOD1 is low MW loading control protein and is stable at high temperatures, could
be used as loading control, during western blot CETSA analysis of proteins which show thermal shift at high

temperatures.

Table S2. Observed temperatures of loading controls under TSPO in cell CETSA conditions.

Figure S3. TSPO thermal curves in HEK cells showing the compound effect of cellular active Ro5 on TSPO at 50 µM
compared to DMSO control sample. Probing with anti-SOD antibody shows that compound addition has no
general effect on protein levels.
Figure S4. Behavior of SERCA2 in the presence of Thapsigargin (10 µM), replicates 2 and 3.

Figure S5. Behavior of SERCA2 in the presence of CDN1163 (10 µM), replicates 2 and 3.

Figure S6. The expression of PAR2 in 1321N1hPAR2 cells without (-) and with (+) PNGase treatment.
Figure S7. CETSA PAR2 thermal shifts in the presence or absence of AZ8838 at 50 µM in 1321N1hPAR2 cells
before PNGase treatment.

Figure S8. CETSA PAR2 thermal shifts in the presence or absence of AZ8838 at 50 µM in 1321N1hPAR2 cells
after PNGase treatment.