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HeLa cells were cultured in DMEM with 10% fetal calf serum and transiently transfected with V5-tagged WT or mutant forms of WDR62 along with AURKB-3xMyc using Lipofectamine (Life Technologies, NY). 36 hours after transfection cells were treated with 0.2 μg/ml nocodazole (Sigma) for 12 hours, then were lysed using a Triton-X100 based buffer containing protease and phosphatase inhibitor cocktails (Calbiochem, San Diego, CA). Clarified supernatants were used for immunoprecipitation using Dynabeads (Life Technologies) according to the supplier’s instructions. Immunoprecipitates were analyzed by Western analysis using standard protocols. In brief, lysates were separated on gradient SDS-PAGE gels (4–16%, BioRad) and transferred to nitrocellulose or PVDF membranes. After blocking with 5% milk, the membranes were incubated first with rabbit anti-tag antibodies and then with a secondary HRP-conjugated anti-rabbit IgG (Jackson Immunoresearch Labs). Signals were detected with chemiluminescence reagent (BioRad).

Human monocyte‐derived macrophages were washed 2× with 1× PBS (Fisher) and stored in −80°C until further analysis. Frozen cell lysates were washed, and lysis buffer comprised of RIPA buffer with protease inhibitor; PMSF (Sigma) was added to the wells to obtain cell lysates. After which, cell lysates were equalized for loading into a gradient SDS–PAGE gels (Bio‐Rad) using the BCA Protein Assay Kit (Pierce). Proteins were separated by Tris–Hcl gel electrophoresis (Bio‐Rad) and transferred onto PVDF membranes (Bio‐Rad). Later, membranes were blocked overnight with blocking buffer (5% milk in 1× PBS‐T with 0.1% Tween‐20). For primary incubation, antibodies against IL‐1β and β‐actin (Appendix Table S1) were incubated for an hour, followed by another 1‐h incubation with HRP‐coupled secondary antibody 1:2,000 (Cell Signaling). Membranes were developed using Amersham^® ECL Western Blotting Detection Reagents (GE Healthcare) following manufacturer's instructions. Quantification of protein band density was performed using ImageJ (NIH) and normalized to β‐actin.

Western blotting was performed as described before.^{22 (link)} Briefly, protein extracts of mouse corneas were prepared in a radioimmunoprecipitation (RIPA) buffer supplemented with 1% SDS and Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). Four corneas were pooled and considered one biological replica. Protein concentration was determined by Bradford-based protein assay (Bio-Rad protein assay). Equal amounts of lysates (30 μg protein) were subjected to electrophoresis in 4% to 15% gradient SDS-PAGE gels (Bio-Rad, Hercules, CA, USA). Protein blots of the gels were blocked with Odyssey blocking buffer (OBB; Li-Cor Biosciences, Lincoln, NE, USA) and incubated overnight with primary antibodies: rabbit anti-α-SMA (ab5694, 1:2000 dilution in OBB; Abcam, Cambridge, MA, USA), goat anti-connective tissue growth factor (CTGF) (clone L-20, 1:500 dilution in OBB; Santa Cruz Biotechnology, Dallas, TX, USA), and mouse anti-β-actin (clone AC-15, 1:10,000 dilution in OBB; Santa Cruz Biotechnology). The secondary antibody used was anti-rabbit IgG IRDye 800CW, anti-goat IRDye 800CW, and anti-mouse IgG IRDye 680LT (1:10,0000 dilution in OBB; Li-Cor Biosciences). Blots were then scanned with the Odyssey Infrared Imaging System, and relative band intensity was quantified by Image Studio v2.0 software (Li-Cor Biosciences).

Cell monolayers were lysed in Mammalian Protein Extraction Reagent (MPER™, Thermo Scientific) supplemented with Halt™ Phosphatase Inhibitor Cocktail (Thermo Scientific) and Complete™ Mini Protease Inhibitor Cocktail (Roche Diagnostics, Indiannapolis, IN). Protein concentrations of cell lysates were determined using the BCA Protein Assay (Thermo Scientific) and proteins were resolved using 7.5%, 12%, or 4-20% gradient SDS-PAGE gels (Bio-Rad, Hercules, CA). Proteins were transferred to supported nitrocellulose membrane overnight at 25 V, and membranes were blocked in 5% non-fat dry milk or 5% BSA in TBST, incubated overnight at 4 C with primary antibody in block, followed by Immun-Star HRP-conjugated secondary antibody (Bio-Rad) at 1:10,000; signal was detected with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Antibodies directed against the following were used: p-DNA-PKcs^T2609 (Thermo Scientific), DNA-PKcs (Santa Cruz Biotechnology, Dallas, TX); AURKA, BRCA1, PARP (Cell Signaling, Danvers, MA); BRCA2 (R & D Systems, Minneapolis, MN), pADPr or PAR (BD Biosciences, San Jose, CA), pH2AX^S139 (Calbiochem), and β-Actin (Sigma-Aldrich). Pixel densities of blot images were calculated using Image-J software (NIH). Changes in protein levels were normalized to loading controls and expressed as fold change relative to treatment controls.

Protein lysates were prepared by cold lysis of 5 × 10⁶ cells in 1× RIPA buffer for 15 min and centrifugation at 12,000 × g and 4 °C for 10 min. Total protein concentration was measured by BCA assay (Pierce), and equal amounts of protein were denatured in 1× LDS buffer with 1× reducing agent (Life Technologies) at 70 °C for 10 min. Samples were separated on 4–15% gradient SDS-PAGE gels (Biorad), blotted onto PVDF membrane, blocked with 3% dry milk in 1× PBS/0.1% Tween 20 (Sigma–Aldrich) and incubated with mouse anti-beta-Actin IgG (1:20,000, Sigma) or rabbit anti-Dicer IgG (1:1000, Sigma–Aldrich) at 4 °C over night. Detection was performed with the IR-Dye system on an Odyssey scanner (Licor) after incubation with anti-mouse (1:10,000) or anti-rabbit (1:5000) secondary antibodies for 60 min at room temperature. Western blot images were analyzed with ImageJ software (Abramoff et al., 2004 ).

Cell lysates were separated by gradient SDS-PAGE gels (Biorad) followed by western blotting with 1 hour of blocking (5% BSA in 0.1% Tween in TBS) at room temperature (RT), overnight primary antibody incubation at 4 °C, and three washes of 10 minutes each at RT. Blots were then incubated with secondary antibody for 1 hour at RT and final three washes of 10 minutes each at RT. Immunoblots were developed using Pierce Pico ECL (Thermo Scientific). Antibodies used were polyclonal phospho-MLC of Thr18/Ser19 (CST), polyclonal MLC (CST) and monoclonal α-tubulin (Sigma).

Whole cell extracts and western blotting was carried out using standard methods. Briefly, total Cell extracts were prepared in 2x Laemmli sample buffer (4% SDS, 20% Glycerol, 120 mM Tris-HCl pH 6.8, 1x beta-mercaptoethanol, 0.002% bromophenol blue). 30–40 μg of extract was loaded into gradient SDS-PAGE gels (Bio-Rad) and transferred to nitrocellulose membranes (Amersham Proton 0.2 μM NC #10600006) blocked in 3–5% skimmed milk TBST and probed with the antibodies listed in Supplementary Data 5. Membranes were developed using either ECL or ECL Prime (Amersham RPN2106/RPN2232) and exposed to Hyblot-ES Autoradiography Film (Denville, #1156p38). Independent Nbs1^f/fISG15^−/− clones were used for each repeat.