Complete protease and phosphatase inhibitor cocktail

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.

Cells were lysed in RIPA buffer (Thermo Scientific, Pittsburg, PA) with complete protease and phosphatase inhibitor cocktails (EMD Millipore, Billerica, MA) and equal proteins were separated on 4–12% Tris-Glycine gel (Biorad Laboratories) under reducing conditions. Proteins were transferred to nitrocellulose membranes, and probed with primary antibodies and appropriate secondary HRP-conjugated antibodies. Immunoreactive proteins were visualized by an enhanced chemiluminescence reagent (Thermo Scientific) and photographed in the Chemidoc (Biorad Laboratories). The antibody sources are: anti-DDR1 (Santa Cruz Biotechnology, Dallas, TX), anti-Vinculin (Millipore, Billerica, MA), and anti-GAPDH (Santa Cruz Biotechnology, Dallas, TX). All secondary antibodies were purchased from Jackson Immunoresearch Lab.

Cells were lysed in RIPA buffer containing a complete protease and phosphatase inhibitor cocktail (Sigma-Aldrich, MO, USA). The protein concentration of the cell lysates was quantified by a BCA Protein Assay Kit (Pierce, Rockford, IL, USA). The same protein samples were resolved onto 10% SDS-PAGE and then transferred onto PVDF membranes (Millipore Q, Billerica, MA, USA). After blocking with 5% nonfat milk at 37℃ for two hours, the membranes were incubated with primary antibodies overnight at 4℃, followed by incubation with the HRP-conjugated secondary antibody for two hours at room temperature. GAPDH antibody was used as a loading control. Finally, the protein band images were captured by a Gene Detection System with an ECL reagent (Thermo Fisher Scientific, MA, USA). The primary antibodies used in the experiments were rabbit polyclonal anti-FAP antibody (GeneTex Inc., CA, USA), rabbit polyclonal anti-lumican antibodies (Absin, Shanghai, China), rabbit polyclonal anti-Serpin E1, anti-Vimentin, and anti-Urease B antibody (Abcam, Cambridge, UK), rabbit monoclonal anti-GAPDH antibody (CST, MA, USA), and rabbit polyclonal anti-α-SMA antibody (Proteintech, Chicago, USA).

After transfection with miRNA, cells were harvested and washed with cold phosphate buffered saline (PBS), and then subjected to lysis by RIPA buffer [Tris-HCl pH 8.0, NaCl, NP-40, Sodium deoxycholate, SDS, Na₃VO₄, EDTA] containing the complete protease and phosphatase inhibitor cocktail (Sigma-Aldrich, Germany). The protein concentration was determined using a micro-BCA protein assay kit (Thermo Fisher Scientific, USA). Cell lysate (40 μg total protein) were separated on SDS-PAGE gel and transferred to polyvinylidene difluoride PVDF membranes (Roche Applied Science, Germany). The membranes were first blocked with 5 % skim milk/Tris-buffered saline containing 0.1 % Tween-20 (TBST) at room temperature (RT) for 30 minutes, after which the membranes were incubated overnight at 4 °C. Membranes were incubated with Primary antibodies against NAMPT (1;1000), and GAPDH (1:3000) overnight at 4 °C followed by incubation with HRP-conjugated anti-rabbit secondary antibody (1:10,000 dilution) for 1 h at RT. The membranes were then washed 3-6 times with TBST and the bands were visualized using an ECL Western blotting detection system (GE Amersham, London, UK). ImageJ software (NIH, Bethesda, MD, USA) was used for the densitometric evaluation of protein bands. The results were normalized to the GAPDH band intensity as internal control.