Anti gapdh gtx100118

40µg total protein were probed by western blot using primary Ab anti-IL-36γ TA505994; Origene, MD; 1:4,000), anti-GAPDH (GTX100118; GeneTex, CA; 1:1,000), and secondary anti-mouse-HRP (7076P2; Cell Signaling, MA; 1:5,000).

Our western blot method has been described elsewhere [34 (link), 35 (link)]. Briefly, the cells were harvested, and total protein was extracted using a urea-based lysis buffer, as described previously. Proteins were subjected to gel electrophoresis and then transferred onto polyvinylidene difluoride membranes. The membranes were incubated overnight at 4°C or at room temperature for 1 hour with the following primary antibodies: anti-Brg1, 21634-1- AP (1:1000, Protein Tech), phospho-Gsk3β (Ser9) (Cell Signaling Technology #9336), anti-Rb, sc-73598 (1:200, Santa Cruz Biotechnology), anti-p21, 2947P (1:1000, Cell Signaling Technology), anti-cyclin D1, 2978P (1:1000, Cell Signaling Technology), anti-Cdk2, GTX22363 (1:200, GeneTex) and anti-Gapdh, GTX100118 (1:1000, GeneTex). The pRb antibodies used in the western blot are the same as those described in the above section. The membranes were then incubated for 1 hour at room temperature with the appropriate biotinylated anti-mouse or anti-rabbit secondary antibody, which were used at a dilution of 1:2000 (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Proteins were detected with a Chemiluminescence Western Bright ECL kit (K-12045-D50, Advansta Inc., Menlo Park, CA, USA).

Ham’s F12 medium, Dulbecco’s Modified Eagle’s Medium (DMEM) (high glucose), fetal-bovine serum (FBS; heat inactivated), L-glutamine, HEPES, insulin-transferrin-selenite (ITS), estrogen, hydrocortisone, penicillin-streptomycin solution, and sodium pyruvate solution was obtained from Gibco (Gaithersburg, MD, USA). The AhR agonists, TCDD and CH223191, were obtained from Sigma-Aldrich (St. Louis, MO, USA). IP was purchased from Supelco^® and FICZ was obtained from Enzo Life Sciences, Inc., Farmingdale, NY, USA. Gelatin from porcine skin and casein was purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA). For western blotting, the following antibodies were utilized: anti-E-cadherin (#3195), β-catenin (#8480), anti-rabbit IgG–HRP (#7074) and anti-mouse IgG–HRP (#7076). All of these compounds were purchased from Cell Signalling (Cell Signalling Technology, Danvers, MA, USA), while anti-Vimentin (GTX00619) and anti-GAPDH (GTX100118) were purchased from Gene Tex (GeneTex, Irvine, CA, USA). Anti-Fibronectin (AB1954-25UL) was obtained from Merck (Darmstadt, Germany).