Total erk t erk

Immunoblotting was the same as previously described [26 (link)]. The following primary antibodies were used: TXNIP (Novus Biologicals), phospho-ERK1/2 (p-ERK; Cell Signaling, Denver, MA, USA), total ERK (t-ERK; Cell Signaling), phospho-JNK (p-JNK; Cell Signaling), total JNK (t-JNK; Cell Signaling), phospho-p38 (p-p38; Cell Signaling), total p38 (t-p38; Cell Signaling), AP-1 (Abcam), and β-actin (Cell Signaling). The relative protein expression was determined using ChemiDoc (Bio-Rad Laboratories).

The total cellular proteins were extracted from treated keratinocytes with lysis buffer (1.5% SDS, 0.0625 M Tris–HCl and 1 mM Na₃VO_4, pH 6.8) containing a protease inhibitor cocktail (Roche, Mannheim, Germany). For Western blotting analysis, 40 μg of extracted total cellular protein was subjected to 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis. After blocking and washing, the membrane was incubated with antibodies including phosphorylated extracellular signal-regulated kinases (pERK), total-ERK (tERK), and GAPDH (Cell Signaling, Beverly, MA), followed by incubation with horseradish peroxidase (HRP)-labelled secondary antibody (1:5000; Jackson Immuno Research, West Baltimore Pike, PA) and developed using a commercial HRP substrate (Immobilon™ Western Chemiluminescent HRP Substrate; Millipore Corporation, Billerica, MA). The membranes were detected by ChemiDoc™ XRS (Bio-Rad Laboratories Inc., Hercules, CA).