Ibright western blot imaging system

Total cell lysates were prepared using lysis buffer (Biosource International) supplemented with Phosphatase Inhibitor Cocktail 2 (P5726, Sigma Aldrich), Phosphatase Inhibitor Cocktail 3 (P044, Sigma Aldrich), Protease Inhibitor Cocktail (Sigma-Aldrich) and PMSF serine protease inhibitor (Sigma-Aldrich), and 10 µg of proteins were separated by SDS-PAGE using Bolt 4–12% Bis–Tris Plus gels (Invitrogen). Gels were transferred onto nitrocellulose membranes using Power Blotter Select Transfer Stacks and the Power Blotter System (Invitrogen). Membranes were blocked with I-Block reagent (ThermoFisher) and incubated with the following primary antibodies overnight at 4 °C: mouse monoclonal anti-calnexin (sc-23954, Santa Cruz); mouse monoclonal anti-CD63 (ab213090, abcam); mouse monoclonal anti-CD81 (ab59477, abcam); mouse monoclonal anti-GAPDH (GTX627408); mouse monoclonal anti-Cytochrome C (SC-13156); mouse monoclonal anti-HSP70/HSC70 (W27, SC-24). Membranes were then incubated with the appropriate peroxidase-conjugated secondary antibody, Goat anti-Mouse (G-21040). Images were acquired using Westar Hypernova ECL substrate (Cyanagen) and iBright Western Blot Imaging Systems (Invitrogen).

For Western blot analysis of XPA protein in skin samples, lysates were prepared using 0.05 mL of RIPA buffer containing 20 mM HEPES, pH 7.4, 2 mM EDTA, 250 mM NaCl, 0.1% NP-40, 2 mg/mL leupeptin, 2 mg/mL aprotinin, 1 mM PMSF, 0.5 mg/mL benzamidine, 1 mM dithiothreitol, and 1 mM sodium vanadate. About 50–80 µg protein was loaded in each well and resolved on 10–12% SDS-polyacrylamide gel and transferred onto PVDF membranes. Membranes were incubated in blocking buffer for 1h and then incubated with the primary antibodies in blocking buffer for overnight at 4 °C. The membrane was then washed with TBS-T and incubated with secondary antibody conjugated with horseradish peroxidase. Protein bands were visualized using the enhanced chemiluminescence detection system (iBright Western Blot imaging systems, Thermofisher Scientific, Waltham, MA, USA). To verify equal protein loading and transfer of proteins from gel to membrane, the blots were stripped and reprobed for β-actin [18 (link),20 (link)]. The band density was analyzed using IMAGE J software provided by the NIH and the values were normalized to the β-actin band density. All the whole western blot figures can be found in the supplementary Figure S1

For the Western blot analysis of different proteins in skin samples, lysates were prepared using 0.05 mL of RIPA buffer containing 20 mM HEPES, pH 7.4, 2 mM EDTA, 250 mM NaCl, 0.1% NP-40, 2 mg/mL leupeptin, 2 mg/mL aprotinin, 1 mM PMSF, 0.5 mg/mL benzamidine, 1 mM dithiothreitol, and 1 mM sodium vanadate. About 50–80 µg protein was loaded in each well and resolved on 10–12% SDS-polyacrylamide gel and transferred onto PVDF membranes. Membranes were incubated in a blocking buffer for 2 h and then incubated with the primary antibodies in a blocking buffer for 2 h at room temperature or overnight at 4 °C. The membrane was then washed with TBS-T and incubated with a secondary antibody conjugated with horseradish peroxidase. Protein bands were visualized using the enhanced chemiluminescence detection system (iBright Western Blot imaging systems, Thermofisher Scientific, Waltham, MA, USA). To verify the equal protein loading and the transfer of proteins from gel to membrane, the blots were stripped and reprobed for GAPDH [38 (link)]. The band density was analyzed using IMAGE J software provided by the NIH and the values were normalized to the GAPDH band density.

The protein concentrations were measured using the BCA Protein Assay kit (Thermo Fisher Scientific). Protein lysate was separated by gel electrophoresis on 10% SDS-polyacrylamide gels. Proteins were transferred to a PVDF membrane. The membranes were blocked for 1 hour at room temperature with 10% blotting-grade blocker (BioRad) in Tris-buffered saline (10 mM Tris, 150 mM NaCl, 0.1% Tween-20 pH 7.5) (TBST) followed by incubation with mouse anti-NKX6.1 (1:5000; DSHB) diluted in blocking buffer, overnight at 4 °C. After intensive washing (minimum 3 times) with TBST (5 min each time), the membranes were incubated with HRP-conjugated secondary antibody (1:10,000 diluted in blocking buffer) for one hour at room temperature, followed by intensive washing with TBST. The protein bands were visualized by iBright Western Blot Imaging Systems (Thermo Fisher Scientific).

One-hundred micrograms of proteins were used for the analysis of 40 cytokines (Eotaxin, Eotaxin-2, GCSF, GM-CSF, ICAM-1, IFN-gamma, I-309, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-6, IL-6sR, IL-7, IL-8, IL-10, IL-11, IL-12p40, IL-12p70, IL-13, IL-15, IL-16, IL-17, IP-10, MCP-1, MCP-2, M-CSF, MIG, MIP-1α, MIP-1β, MIP-1delta, RANTES, TGF-β1, TNF-α, TNF-β, sTNF RI, sTNF-RII, PDGF-BB, TIMP-2) using the Human Inflammation Antibody Array-Membrane Kit (ab134003; Abcam, Cambridge, UK) following the manufacturer’s instructions. In brief, the array membranes were blocked by incubation with a blocking buffer for 30 min. After incubation, membranes were incubated with biotin-conjugated anti-cytokines overnight at +4 °C and then with HRP-Conjugated Streptavidin for 2 h. The signal was detected by chemiluminescence with iBright Western Blot Imaging Systems (Thermo Fisher Scientific-Thermo Fisher). Quantification of each protein spot was performed by measuring integrated density using densitometry Image J v3.91 software.

Immunoblotting was performed as described earlier with some modification [16 (link),17 (link),18 (link)]. Briefly, whole-cell lysates were prepared from 2D or 3D cultures. Cells and spheroids were washed with cold 1X PBS and lysed using RIPA lysis buffer (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% TritonX-100, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM Phenylmethylsulfonyl fluoride (PMSF), 4 μg/mL aprotinin, 4 μg/mL leupeptin, 0.6 μg/mL benzamidinchloride, 20 μg/mL trypsin inhibitor) for 15 min at 4 °C. Lysates were centrifuged at 4 °C for 15 min at 13,000 rpm to remove insoluble debris. Protein concentration in the lysate was estimated using bicinchoninic acid (BCA method) as described by the manufacturer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Proteins were then resolved on a 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane (Millipore Corporation, Burlington, MA, USA). The PVDF membrane was blocked with 5% nonfat dry milk and was incubated with primary antibody (1:1000 dilution) overnight at 4 °C followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Bio-Rad, Hercules, CA, USA). β-actin was used as a loading control. The expression of specific proteins was detected using chemiluminescence in iBright Western Blot Imaging Systems (Thermofisher scientific, Waltham, MA, USA). For antibodies, see Table S2.