Anti rabbit hrp secondary antibodies

The ethanolic extract of A. camphorata was provided by Biotech Lantyng Company (Taipei, Taiwan). Cell culture reagents were obtained from Gibco (USA). GW9662, Rosaglitazone, and MTT were purchased from Sigma (USA). PPAR-γ (D69), ki-67 (D3B5) mAb, Caspase-3, β-actin, α,β-Tubulin and GAPDH antibodies, and anti-rabbit-HRP secondary antibodies were obtained from Cell Signaling Technology (USA), while GLK, p-IRE1α (phospho-S724), IRE1α antibodies were obtained from Abcam (USA). p-PERK (phosphor-Thr981), PERK (aa947-996) antibodies were purchased from LifeSpan BioSciences (USA), while GLUT-2 (H-67) and ATF6α (H-280) antibodies were purchased from Santa Cruz Biotechnology (USA).

Total protein lysates were prepared from pooled untreated or treated keratinocytes using RIPA buffer containing Halt protease/phosphatase inhibitors in accordance with the manufacturer’s protocol (Thermo Fisher Scientific, Rockford, IL, USA). 25 μg of protein was electrophoresed using 4–12% gradient SDS-polyacrylamide gels, transferred to PVDF membrane and blocked with 5% BSA. Primary antibodies for pEGFR/EGFR, MET, pHER2/HER2, pHER3/HER3, pAKT/AKT, STAT3, pmTOR/mTOR, B-Actin were purchased from Cell Signaling and used at 1:1000. p-MET was purchased from Abcam and used at 1:1000. Anti-rabbit HRP secondary antibodies (Cell Signaling Technology), followed by West Dura Chemiluminescence substrate (Thermo, Rockland, IL) were used for signal detection. Bands were quantified using NIH Image J and normalized to the densitometry for the respective housekeeping gene. Western blots used pooled keratinocyte protein (n = 4–6 mice/pool) and were repeated a minimum of three times.

Total protein lysates were prepared from keratinocytes and fibroblasts using RIPA buffer containing Halt protease/phosphatase inhibitors in accordance with the manufacturer’s protocol (Thermo Fisher Scientific, Rockford, IL, USA). Twenty-five micrograms of protein was electrophoresed using 4–12% gradient SDS-polyacrylamide gels, transferred to PVDF membrane and blocked with 5% BSA. Primary antibodies for HGF (Abcam; cat #ab83760), TGFβ (Abcam; cat #ab92486), TGFβR1 (Abcam; ab31013), TGFβR2 (Abcam; ab186838), SMAD2 (Abcam; ab63576), SMAD4 (Abcam; ab236321) MET (Cell Signaling; cat #3127), p-MET (Abcam; ab5662), GAPDH (Cell Signaling, Cat #5174), B-actin (Cell Signaling; Cat #4970), tubulin (Cell Signaling, Cat #15115) were incubated with membranes at 1:1000. Anti-rabbit HRP secondary antibodies (Cell Signaling Technology), followed by West Dura Chemilluminescent substrate (Thermo, Rockland, IL) were used for signal detection. Bands were quantified using NIH Image J and normalized to the densitometry for the respective housekeeping gene. All Western blots used pooled protein from triplicate samples and were repeated a minimum of three times. Quantification of Western blots is displayed in Supplementary data A.