P erk thr202 tyr204

Lysates from whole skin, epidermis, dermis or cultured cells were prepared as previously described^{7 (link)} using buffers supplemented with Complete protease and phosphatase inhibitor cocktails (Merck). Protein concentrations were measured using Bradford reagent (BioRad, Hercules, California, USA) and 20–30 µg of protein/sample was boiled in Laemmli buffer, separated on SDS-PAGE, and transferred to Hybond ECL nitrocellulose (GE Healthcare, Illinois, USA). Nitrocellulose membranes were stained with Ponceau S (Merck) to verify equal protein loading and transfer prior to blocking and antibody incubations. Primary antibodies used were from Cell Signalling Technology (Danvers, Massachusetts, USA): p-GR Ser²¹¹, #4161S, 1/2000; p-ERK Thr²⁰²/Tyr²⁰⁴, #4376, 1/1000; p-p38 Thr¹⁸⁰/Tyr¹⁸² #4631, 1/1000; p-JNK Thr^183/Tyr¹⁸⁵ #9251, 1/1000, and JNK #9252, 1/1000; Santa Cruz Biotechnology (Dallas, Texas, USA): GR, sc1004, 1/2000; Sigma: actin, A2066, 1/4000; and tubulin, T6199, 1/4000. Peroxidase-conjugated secondary antibodies were from GE Healthcare: anti-rabbit, NA934 and anti-mouse NXA931. Immunoreactive bands were detected using Pierce ECL Plus Western Blotting Substrate (ThermoFisher) and the ImageQuant 4000 Biomolecular Imager (GE Healthcare). Band intensities were quantitated using Image J software and were normalized to the loading controls, actin, or tubulin.

Whole cell lysates were prepared using EBC lysis buffer (50 mM Tris–HCl [pH 8.0], 120 mM NaCl, 1 % Triton X-100, 1 mM EDTA, 1 mM EGTA, 0.3 mM phenylmethylsulfonyl fluoride, 0.2 mM sodium orthovanadate, 0.5 % NP-40, and 5 U/mL aprotinin) and centrifuged. Proteins were separated using SDS-PAGE and transferred to PVDF membranes (Invitrogen) for western blot analysis. Membranes were probed with antibodies against p-ALK (Tyr1604), ALK, p-Akt (Ser473), P-gp (all from Cell Signaling Technology, Beverly, MA), Akt, p-Erk (Thr202/Tyr204), Erk, HSF1, Hsp90, Hsp70, Hsp27, NQO1, and β-actin (all from Santa Cruz Biotechnology, Santa Cruz, CA) as the first antibody, and then membranes were treated with horseradish peroxidase-conjugated secondary antibody. All membranes were developed using an enhanced chemiluminescence system (Thermo Scientific, Rockford, IL).