Anti twist 1 sc 81417

Immunofluorescence and immunohistochemistry assays were performed as previously described [2 (link), 10 (link)]. The following primary antibodies were used: anti-Cx43 (Sigma-Aldrich, C6129), anti-collagen II (Invitrogen, Thermo Fisher Scientific, MA5-12789), anti-Ki-67 (BD, 550609), anti-Twist-1 (sc-81417) and anti-NF-κB (sc-8008) from Santa Cruz Biotechnology. Goat anti-rabbit FITC-conjugated (F-2765, Invitrogen, Thermo Fisher Scientific) and goat anti-mouse Alexa 594-conjugated (A-11032, Invitrogen, Thermo Fisher Scientific) secondary antibodies were used.

Protein extracts from cells were obtained as previously reported [17 (link)]. The protein concentrations were determined using the Bradford assay [52 (link)]. Protein extracts (25 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), incubated with anti-Twist1 (SC-81417, Santa Cruz Biotechnology), anti-IL-17RA (FAB177P, R&D Systems, Minneapolis, MI, USA), and anti-Act1 (SC-100647, Santa Cruz Biotechnology) antibodies at a 1:500 dilution at 4 °C overnight, followed by incubation with the appropriate secondary antibody at a 1:2000 dilution at room temperature for 2 h. Anti-GAPDH (SC-47724, Santa Cruz Biotechnology) at a 1:2000 dilution was used as loading control. Antibody binding was detected using enhanced chemiluminescence with Pierce Plus Western Blotting Substrate (Thermo Scientific). Images were obtained using iBright FL1500 Imaging System instrument (Thermo Scientific). Densitometric analysis was conducted using ImageJ 1.53k (NIH). Values obtained for GAPDH staining was used to normalize the densitometry values to the probed targets.

Cell lysates were harvested using cold RIPA buffer (#R0278; Sigma‐Aldrich) with protease inhibitor (#539134) and phosphatase (#524625) inhibitor cocktails from Calbiochem, Millipore (Burlington, MA, USA). BCA assay (#23225; Thermo Scientific, Waltham, MA, USA) was performed for protein quantification. After heating, lysates were resolved by standard reducing SDS/PAGE, transferred to poly(vinylidene difluoride) membranes, blocked with 5% BSA for at least 1 h, and incubated overnight at 4 °C with the following antibodies diluted in 1% BSA in TBST: anti‐E‐cadherin (#610182) from BD Biosciences, Franklin Lakes, NJ, USA at 1 : 1000; anti‐TWIST1(#sc‐81417) from Santa Cruz Biotechnology, Dallas, TX, USA at 1 : 200; antivimentin (#M7020) from Agilent Technologies (Dako, Santa Clara, CA, USA) at 1 : 1000; anti‐actin (#A1978) and anti‐GAPDH (#G9545) from Sigma at 1 : 1000. After washing, membranes were then incubated in dark at room temperature for half an hour with Infrared dye‐conjugated secondary antibodies from Li‐COR Biosciences, Lincoln, NE, USA at 1 : 5000 diluted in TBST: IRDye 800CW goat anti‐mouse or anti‐rabbit (#926‐32210, #926‐32211) and IRDye 680LT goat anti‐mouse or anti‐rabbit (#926‐68020, #926‐68021). Blots were scanned using the Odyssey Infrared Imaging System (Li‐COR). Images were transferred to grayscale.