Imagequant 4000 biomolecular imager

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.

Frozen skin samples were used for protein isolation (whole cell extracts or cytoplasmic and nuclear fractions)^{15 (link)} after dissecting out the adipose tissue, followed by immunoblotting. Briefly, samples (30 μg/lane) were boiled in Laemmli buffer, separated on 8% SDS-PAGE and transferred to nitrocellulose membranes (Hybond ECL, GE Healthcare Bio-Sciences, Pittsburgh, PA). Then, membranes were blocked with 5% nonfat dry milk in PBS-0.1% Tween 20 and incubated with specific primary antibodies overnight at 4 °C. After washing, membranes were incubated with secondary peroxidase-conjugated antibodies, washed again and signal was detected with ECL2 (Thermo Scientific) and the ImageQuant 4000 Biomolecular Imager (GE Healthcare). Band intensities were quantitated using ImageJ software. All signals were normalized to the indicated controls (actin for whole cell extracts, or tubulin and laminA/C for cytoplasmic and nuclear extracts, respectively) with a minimum of three biological replicates. The GR activity was measured as a ratio of pGR/GR^{14 (link)}.

All protein extraction buffers were supplemented with Complete protease and phosphatase inhibitor cocktails (Roche). Whole cell protein extracts from cultured keratinocytes were prepared using RIPA buffer and those from mouse tissues were prepared by pulverizing frozen tissue, then subjecting it to 3 freeze thaw cycles in 20 mM HEPES pH7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EDTA, 25% glycerol, then adding IGEPAL® CA-630 NP-40 to a final concentration of 1%. Nuclear cytoplasmic fractionation was carried out as described^{41 (link)}. Samples (25–30 μg/lane) were boiled in Laemmli buffer, separated on SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare). Membranes were blocked and incubated with specific primary antibodies then peroxidase-conjugated secondary antibodies following manufacturers’ indications. Immunoreactive signal was detected with Pierce ECL Plus Western Blotting Substrate (ThermoFisher) and the ImageQuant 4000 Biomolecular Imager (GE Healthcare, Little Chalfont, UK). Band intensities were quantitated using Image J software. All signals were normalized to loading controls (tubulin, actin or laminA).