Imagequant las 4000

The cells from different groups were lysed in protein lysis buffer (Intron Biotechnology) containing various protease inhibitor cocktail and PMSF (1 mM). After centrifugation of the lysate at 4 °C, the supernatant was collected and used for the determination of protein concentration using a Bradford assay. The cells lysates containing equal amounts of proteins (5–25 µg) was resolved using an 8–12% SDS-polyacrylamide gel electrophoresis and the proteins were electrotransferred to polyvinylidene fluoride (PVDF) membranes. The membrane was blocked with 5% skimmed milk for 30 min in Tris-buffered saline (TBS; Boster Biological Technology, Ltd., Wuhan, China), and then incubated in appropriate primary antibodies in 1% skimmed milk in TBS containing 0.1% Tween20 (TBST) overnight at 4 °C. The membrane was washed three times in TBST, and then incubated with the corresponding secondary antibodies diluted in 1% skimmed milk in TBST (1:1000–1:5000) for 1–2 h at room temperature. Enhanced chemiluminescence detection system (Amersham Pharmacia Biotech) and Image Quant Las-4000 were used for detection of antibody binding. Antibodies for β-actin, JNK, p-JNK, ERK, p-ERK, p38, MMP-9, and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-MMP-2 and anti-p-p38 antibodies were purchased from Cell Signaling (Beverly, MA, USA).

Western blot analysis of indigenous MBL was performed on loading various whole cell lysates onto a Tris-HCl polyacrylamide gel (15%). Proteins were transferred to a PVDF membrane with Mini Trans-Blot (Bio-Rad Laboratories, Inc., USA) at constant current 400 mA for 2 h. The protein-transferred membranes were blocked with skin milk (5%)/TBST (50 mM Tris 150 mM, NaCl 0.05%, Tween 20, pH 7.6) for 1 h and probed with a rabbit polyclonal antibody (anti-MBL), or anti-6His antibody overnight at 4°C on a rocking platform. Membranes were then washed in TBS-0.1% Tween 20 five times. The horse-Radish peroxidase-conjugated anti-IgG antibody (from rabbit) served as the secondary antibody to probe the primary antibody on the PVDF membrane. Western blotting detection reagent (Amersham ECL Prime) was utilized to develop the signal and monitored with Image Quant LAS 4000 (Amersham, GE Healthcare, USA).

Primary keratinocytes were sequentially transduced with viral vectors encoding v-ras^Ha, and either ΔNp63α or Stuffer, as described above. Three days post-transduction of lentivirus-ΔNp63 or Stuffer, the cell culture medium was replaced with fresh medium; 24 hours later the culture supernatant was collected and immediately incubated with a dot blot antibody array at 4°C overnight (Mouse Cytokine Array C1000, Raybiotech) according to the manufacturer’s instructions. The image was developed using Amersham ImageQuant LAS 4000.

To further characterize brown adipose tissue in mice, we used the Proteome Profiler Mouse Adipokine Array Kit (R & D, Minneapolis, MN, USA) to measure the levels of secreted adipokines in BAT cultured media, using the manufacturer’s instructions. Proteome Profiler membranes were visualized using a ImageQuant LAS 4000 (Amersham, The Netherlands).

Separated proteins on PAGE gels were transferred onto PVDF membranes (0.2 μm) using a Trans-Blot Turbo RTA Transfer Kit (Bio-Rad). Blots were washed for 1 min with TBST followed by blocking with Superblock (Thermo-Fisher) for 30 min. Afterwards, blots were incubated overnight at 4 °C with primary antibodies anti-Strep-tag II (1:2000; NBP2-43735 Novus biologicals) or anti-FLAG (1:2000; F1804 Sigma). Unbound antibodies were removed via three 5 min washes with TBST followed by incubation of the blot for 1 h with the HRP-conjugated anti-mouse secondary antibodies (1:10,000; A9044, Sigma-Aldrich). After three successive washes with TBST and one wash with TBS, Thermo Scientific™ SuperSignal™ West Pico PLUS Chemiluminescent Substrate ECL substrate (Thermo) was added, and signal detection was obtained with an ImageQuant™ LAS 4000 (Amersham).