Novex ecl

Total protein was extracted using cell lysis buffer containing protease and phosphatase inhibitor mixtures. Equivalent protein concentrations were loaded into 4–12% NuPAGE Novex Tris acetate gradient gels (ThermoFisher Scientific, Waltham, MA, USA) for electrophoresis. Proteins were subsequently transferred to a nitrocellulose membrane using a Criterion blotter apparatus (Bio-Rad, Hercules, CA, USA). The membrane was incubated in 5% non-fat dry milk in TBST for 1 h. Immunoblots were incubated overnight with primary antibody in TBST at 4 °C. The next day immunoblots receive 3–5 min washes in TBST before incubation with secondary antibody diluted in TBST for 1 h. Detection of immunoreactive bands was performed using chemiluminescence (Novex ECL, Invitrogen, Waltham, MA, USA). Blots were stripped using Restore Plus Western Stripping Buffer (Thermo Fisher Scientific) for 5–10 min at room temperature and reprobed using another primary antibody. Developed X-ray films were imaged and digitized using a Bio-Rad GelDoc with ImageLab software. Pixel intensities for bands were used for quantification after normalization to loading control bands (α-tubulin). Active Rac1-Pulldown was performed as per manufacturer instructions using Rac1 Pull-Down Activation Assay Biochem Kit (Cytoskeleton Inc., Denver, CO, USA).

Total cell extracts were obtained using a lysis buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 1 mM MgCl₂, 1 mM EDTA, 2 mM EGTA, 10% glycerol, 1% Triton X-100, 1 μg/mL leupeptin, and 1 μg/mL aprotinin). Equal amounts (40 μg) of cell lysates were resolved on 8% or 12% Bis-Tris gels with MOPS running buffer (Invitrogen, Novex), transferred to PVDF membranes, and incubated with specific antibodies against MerTK (Abcamplc, Cambridge, UK) and beta-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Immune complexes were visualized as enhanced chemiluminescence (Novex ECL, Invitrogen).

Fluorescent proteins were visualized directly in the agarose gels using a UV transilluminator (Cleaver Scientific, Rugby, UK). Blotted membranes were stained with Ponceau S solution, and proteins were also detected by means of anti-HisTag-HRP (horseradish peroxidase) antibodies (Thermo Fisher, Cat No. MA1-80218, Waltham, MA, USA) in combination with Novex™ ECL (Invitrogen Chemiluminescent Substrate Reagent Kit, Cat No. WP20005, Waltham, MA, USA) as a reaction substrate. Membranes were imaged with a UVITEC chemiluminescence imaging system (Cambridge, UK). In situ APEX activity in agarose gels was detected in the presence of TMB (3,3′,5,5′-Tetramethylbenzidine) (VWR—Cat. No. J644-100 ML) until colored bands appeared. APEX was used to identify blotted antigen (SC) by adding ACA diluted 1:200 in PBS/5% milk in combination with a Chemiluminescent Substrate Reagent Kit (Invitrogen, Cat No. WP20005). Membranes were imaged as above. APEX activity was also measured on membranes blotted with ACA. Membrane lanes were cut and incubated with either TMB or Amplex^®UltraRed, and APEX activity was measured by using the spectrophotometer to read the absorbance at 450 nm and the fluorescence at 595 nm for TMB and Amplex^®UltraRed, respectively.

Western blot analysis of IRS1, Bcl-2, and GAPDH were performed according to standard procedures. All antibodies were from Cell Signaling Technologies and diluted 1:1000 in 1% BSA. INS-1 cells and pancreatic tissue were lysed in RIPA buffer on ice and protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Equal amounts of protein (20 μg) were electrophoresed through 4% SDS-polyacrylamide stacking gels and 10% SDS-polyacrylamide separating gels and then transferred to PVDF membranes (Millipore, Darmstadt, Germany). After blocking at room temperature in TBST containing 5% BSA, membranes were incubated with primary antibodies against INS-1 and GAPDH overnight at 4°C with gentle shaking. Following three washes in TBST, membranes were incubated with secondary antibodies conjugated to horseradish peroxidase. Signal development was performed with Novex ECL (Invitrogen), films were scanned into TIFF format at 600 dpi resolution, and ImageJ software was used for signal quantification (Heinke et al., 2008).

Proteins of LSC colonies were isolated with a cell extraction buffer (Invitrogen, Carlsbad, CA) supplemented with protease inhibitor cocktail (Roche, Basel, Switzerland). Forty μg total protein provided with loading buffer (Bio-Rad Laboratories, Inc.) and 100 mM dithiothreitol were electrophoretically separated on polyacrylamide gels (Excel-Gel SDS 8–18 Gradient, GE Healthcare, Little Chalfont, UK). For LC3B-I/II immunoblotting, a precast 4–20% gradient gel (Criterion TGX, Bio-Rad Laboratories, Inc.) was used. Proteins were transferred to a nitrocellulose membrane (0.2 μm pore size, Invitrogen). Five % (w/v) nonfat dried milk powder and 2% (w/v) bovine serum albumin (BSA, Sigma-Aldrich) in tris buffered saline containing 0.05% (v/v) Tween20 was used for membrane blocking and as antibody diluent. Peroxidase-conjugated secondary antibodies were visualized using Novex ECL (Invitrogen). Chemiluminescence was detected using ChemiDoc XRS system with Image Lab software (version 5.2.1, Bio-Rad Laboratories, Inc.).

Transfected COS‐7 cells were lysed in RIPA buffer [50 mM Tris–HCl (pH 7.4), 0.15 M NaCl, 0.25% deoxycholic acid, 1% NP‐40, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, phosphatase inhibitor cocktail 2 and 3 (Sigma‐Aldrich), and protease inhibitor cocktail (Sigma‐Aldrich)], followed by centrifugation at 15,000 × g for 12 min. For SDS–PAGE, 15% Phos‐tag precast gel (SuperSep Phos‐tag, Wako) was used. After electrophoresis, Phos‐tag acrylamide gels were washed three times and stirred gently in transfer buffer (Fast buffer, ATTO) containing 0.01% SDS and 10 mM EDTA for 10 min. Then, it was incubated in the transfer buffer containing 0.01% SDS without EDTA for 10 min according to the manufacturer's protocol. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes and immunoblotted with the anti‐Flag (M2; Sigma‐Aldrich) or anti‐GFP (mouse JL‐8; Clontech) antibody. The bound antibodies were visualized with horseradish peroxidase (HRP)‐conjugated antibody against mouse IgG using the HRP chemiluminescent substrate reagent kit (Novex ECL; Invitrogen).

Immunoblots were performed following standard procedures. Primary antibodies used were: SOX9 (AB5535, Millipore), BMI1 (05-637, Millipore), p21^CIP (sc-397-G, Santa Cruz Biotechnology), GFP (ab6673, abcam) and β-actin (AC-15, Sigma). Primary antibodies were detected with HRP-linked secondary antibodies: anti-rabbit (7074S, Cell Signaling Technology), anti-mouse (7076S, Cell Signaling Technology) and anti-goat (sc-2020, Santa Cruz Biotechnology). Protein bands were detected using the ECL system (NOVEX® ECL, Invitrogen).