Anti rabbit secondary antibody

DMEM (high glucose, [4.5 g/L]), NaCl, FBS, and a BCA protein assay kit (Pierce, Rockford, IL, USA) were purchased from Euroclone (Milan, Italy); Na₃PO₄, MgSO₄, NaH₂PO₄, KH₂PO₄, and KCl were purchased from Carlo Erba (Milan, Italy); ferric citrate, Trypsin-EDTA-Solution (T4174) and Collagenasi type IA (C9891) were purchased from Sigma (St. Louis, MO, USA); the primary antibody for Axl (sc-1097) and GAS6 (N-20 sc-1936) were purchased from Santa Cruz (Heidelberg, Germany); the primary antibody for LC3-II was purchased from Cell Signalling (Danvers, MA, USA), and the anti-rabbit secondary antibody was purchased from GeneTex (Irvine, CA, USA); the anti-goat secondary antibody (ab6741) was purchased from Abcam (Cambrige, UK); Hepes Buffer Solution, PVDF membrane Invitrogen/Applied Biosystem (Milan, Italy); PE Annexin V Apoptosis Detection Kit I (559763) was purchased from Bioscience. An ApopTag Red In Situ Apoptosis Detection kit S7165 was purchased from Chemicon. All other reagents were obtained from Sigma (St. Louis, MO, USA).

Antibodies used in this study, including anti-actin, anti-PKA, anti-GSK3β, anti-p-GSK3β, anti-mouse, and anti-rabbit secondary antibody were purchased from GeneTex (Irvine, CA, USA). Antibodies including anti-p-ERK and anti-CREB were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies including anti-p-CREB, anti-MITF, and anti-p-MITF were purchased from Abcam (Cambridge, UK). Other primary and secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

The cells were lysed with cell lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA). A total of 40 µg of proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were then blocked with 4% of BSA (Morebio, Inc., Hanam, South Korea) in TBS-T (0.1% Tween) in RT for 1 h for ELK3 protein and 2% BSA in TBS-T (0.1% Tween) in RT for 1 h for GAPDH, Fibronectin and E-cadherin protein. The membranes were probed overnight at 4 °C with anti-ELK3 (NBP2-01264; 1:1000, Novus Biologicals, LLC, Minneapolis, MN, USA), anti-E-cadherin (sc-7870; 1:1000, Santa Cruz Biotechnology, Inc. Dallas, TX, USA), anti-Fibronectin (F3648; 1:1000, Sigma-Aldrich) or anti-GAPDH (sc-166574; 1:1000, Santa Cruz Biotechnology, Inc.) antibodies, and then washed with TBS-T (0.1% Tween). After washing, the membranes were incubated for 1 h at room temperature with an anti-mouse secondary antibody (GTX213111-01; 1:4000, GeneTex, Inc., Irvine, CA, USA), or an anti-rabbit secondary antibody (GTX213110-01; 1:4000, GeneTex, Inc.). Immunoreactivity was detected using an ECL kit (Thermo Fisher Scientific, Inc.) and ImageQuant Las 4000 (GE Healthcare, Chicago, IL, USA).