Phospho smad2 ser465 467 smad3 ser423 425

Frozen skin tissue was pulverized using liquid nitrogen, dissolved in Laemmli
buffer and RIPA buffer (1% Triton X-100 in PBS with 10 mM
EDTA, 150 mN NaCl, 1% sodium deoxycholate and
0.1% SDS) and sonicated. Samples were resolved by SDS–PAGE
and probed by immunoblotting. The band intensities in the western blots were
determined by ImageJ software. Immunoblotting was performed with antibodies
against phospho-NFκB p65, NFκB (Cell Signaling,
#3034), α-SMA, Fibulin-5, Smad2/3 (Cell Signaling,
#5678), phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (Cell Signaling,
#8823), FAK (Cell Signaling, #3285), phospho-FAK (Tyr 397, Cell
Signaling, #3283) and tubulin (Sigma-Aldrich, clone B-5-1-2). Primary
antibodies were used at 1:1,000 dilution and horse radish peroxidase-conjugated
secondary antibodies (Jackson Immunoresearch) were used at 1:10,000
dilution.

AMTB was purchased from Merck KGaA company (#SML0103). Cisplatin was purchased from Sigma-Aldrich company (#61825-94-3). The antibody to TRPM8 was bought from Abcam (#ab3243). The antibodies to Caspase-3 (#9662), PARP (#9542), Smad2/Smad3 (#8685) and Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (#8828) were bought from Cell Signaling Technology, Inc. The antibody to β-Actin was bought from Medical & Biological Laboratories Co., LTD. (#M177-3).

Crizotinib was kindly provided by Pfizer. Brigatinib (AP26113; Cat. #S8229) and lorlatinib (PF-6463922; Cat. #S7536) were purchased from Selleckchem (Houston, TX, USA). Silibinin (Cat. #S0417) was purchased from Sigma-Aldrich (Madrid, Spain). All reagents were dissolved in sterile dimethylsulfoxide (DMSO) to prepare 10 mmol/L stock solutions, which were stored in aliquots at −20 °C until use. Working concentrations were diluted in culture medium prior to each experiment.
Antibodies against E-cadherin (#3195), SMAD2/3 (#3102) and phospho-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) (#9510) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against GADPH (#60004-1-Ig) and β-actin (#66009-1-Ig) were purchased from Proteintech Group, Inc (Rosemont, IL, USA). Antibodies against vimentin (#V6630) and SNAIL (#MA5-14801) were purchased from Sigma-Aldrich and ThermoFisher Scientific Inc. (Waltham, MA, USA), respectively.
The Applied Biosystems^TM TaqMan^TM Array Human TGFβ Pathway 96-well Plate (Cat. #4414097) was purchased from Applied Biosystems (Foster City, CA, USA). RayBio^® C-Series Human TGFβ Array C2 (Cat. #AAH-TGFB-2-2) was purchased from RayBiotech, Inc. (Norcross, GA, USA).