P44 42 mapk erk1 2 rabbit monoclonal antibody

Western blotting was performed as described previously (Silva-Peña et al., 2018 (link)). An appropriate combination of primary and secondary HRP-conjugated antibodies was used: Phospho-(Thr202/Tyr204)-p44/42 MAPK (ERK1/2) rabbit polyclonal antibody (1:1,000, Cell Signaling, #9101), p44/42 MAPK (ERK1/2) rabbit monoclonal antibody (1:1,000, Cell Signaling, #4695), phospho-(Thr308)-AKT1/2/3 rabbit polyclonal antibody (1:1000, Santa Cruz, #sc-16646-R), AKT1/2/3 rabbit polyclonal antibody (1:1000, Santa Cruz, #sc-8312), adaptin γ mouse antibody (1:1,000, BD Biosciences, #610385) and HRP-conjugated anti-rabbit or anti-mouse IgG (H+L) secondary antibodies (1:10,000, Promega, Madison, WI, United States). The membranes were incubated for 1 min with the Western Blotting Luminol Reagent kit (Santa Cruz, CA, United States), and the specific protein bands were visualized and quantified by chemiluminescence using a ChemiDoc^TM MP Imaging System (Bio-Rad). Western blots showed that each primary antibody detected a protein of the expected molecular size. The protein intensity was quantified with the image processing software ImageJ (Rasband, W.S., ImageJ, United States, NIH¹, 1997–2012). The results were expressed as the protein/adaptin γ and phosphorylated/total protein ratios.

Cytoplasmic cell extracts were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes for western blotting. Membranes were incubated with p44/42 MAPK (ERK1/2) rabbit monoclonal antibody (Cell Signaling Technology, Danvers, USA), Phospho-p44/42 MAPK (ERK1/2) rabbit monoclonal antibody (Cell Signaling Technology), or anti β-actin antibody (Sigma-Aldrich, St. Louis, USA) at 4°C overnight, as appropriate. After washing with Tris-buffered saline (TBS) containing 0.02% (v/v) Tween 20, membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Sigma-Aldrich) at 37°C for 1 h. Immunoreactions were then visualized using the enhanced chemiluminescence detection system (UVP Inc., Upland, USA).

The following antibodies were used: p44/42 MAPK (ERK1/2) rabbit monoclonal antibody (Cat# 9102, RRID:AB_330744; Cell Signaling Technology, Beverly, MA, USA), phospho-p44/42 MAPK (ERK1/2; Thr202/Tyr204) rabbit monoclonal antibody (Cat# 9101, RRID:AB_331646; Cell Signaling Technology), Akt (pan) (C67E7) rabbit monoclonal antibody (Cat# 4691, RRID:AB_915783; Cell Signaling Technology), phospho-Akt (Ser473) rabbit monoclonal antibody (Cat# 9271, RRID:AB_329825; Cell Signaling Technology), and anti-phospho-AMPKα (Thr172) rabbit monoclonal antibody (Cell Signaling Technology), anti-AMPKα rabbit monoclonal antibody (Cell Signaling Technology), anti-CRBN mouse polyclonal antibody (Abnova, Taipei, Taiwan), anti-AGEs mouse monoclonal antibody (Transgenic, Inc., Kobe, Japan), anti-GAPDH mouse monoclonal antibody (Wako).