Immobilon psq membrane

Cells were lysed in Laemmli sample buffer (Bio‐Rad, Hercules, CA, USA) supplemented with a protease inhibitor complete EDTA‐free (Roche). Protein concentration was measured using BCA Protein Assay kit (Pierce, Rockford, MA, USA). Cell lysates (50 µg) were electrophoresed on 10%‐20% polyacrylamide gels (Bio‐Rad) and transferred to Immobilon PSQ membranes (Millipore, Bedford, MA, USA). The membranes were blocked with TBS containing 5% skim milk and 0.1% Tween‐20, then incubated with the primary antibody overnight, at 4°C. Antibodies for collagen I and collagen III were purchased from Abcam (Cambridge, UK). The membranes were incubated after washing with HRP‐conjugated goat anti‐rabbit IgG (Calbiochem, Gibbstown, NJ, USA) and analysed using enhanced chemiluminescence plus reagent (GE Healthcare, Buckinghamshire, UK).

Total protein extracts were prepared by boiling the cells in SDS sample buffer for 10 min at 95 °C. The proteins were then separated by 10% SDS-PAGE and transferred onto Immobilon-P^SQ membranes (Millipore). Western blotting was performed by incubating the membranes overnight at 4 °C with primary antibodies against the following proteins: Brm (ab15597; Abcam), BRG1 (sc-10768; Santa Cruz), Halo tag (G928A; Promega), HA tag (#3724; Cell Signaling), and β-actin (sc-47778; Santa Cruz). After three washes with Tris-buffered saline containing Tween 20, the membranes were incubated with secondary antibodies [donkey anti-rabbit-horseradish peroxidase (AP182P) or donkey anti-mouse-horseradish peroxidase (AP192P); Millipore] for 1 h at room temperature. Signals were detected using ECL reagent (Promega) or ImmunoStar LD (Wako). Amounts of charged protein samples were roughly normalized to β-actin.

Cells were lysed in Laemmli sample buffer (BioRad) supplemented with a protease inhibitor (Roche). Protein concentration was measured using a BCA Protein Assay kit (Thermo Scientific). Cell lysates (40–45 mg per lane) were electrophoresed on 10–20% polyacrylamide gels (Bio-Rad) and transferred to Immobilon-PSQ membranes (Millipore). The membranes were blocked with TBS containing 5% skim milk and 0.1% Tween-20 (TBST), then incubated with the primary antibody anti-INTS6 (Abcam). The membranes were incubated after TBST washing with HRP-conjugated anti-mouse secondary antibody (Cellsignaling) and analyzed using enhanced chemiluminescent HRP Antibody Detect Reagent (Denvillle Scientific). The online available software ImageJ was used to quantify the density of the bands. The expression of INTS6 and INTS6P1 was normalized to that of beta-Actin (Abcam).

After treatments, cells or tissues were subjected to SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and protein concentrations in supernatants were measured using a Bio-Rad (Hercules, CA, USA) protein assay kit and bovine serum albumin (BSA) as the standard. Proteins in supernatants (17 μg protein per lane) were then separated by 12% SDS-PAGE and transferred to Immobilon-P^SQ membranes (Millipore; MA, USA). Membranes were immediately placed in 5% skim milk for 30 min and then incubated with the following primary antibodies; GFAP (mouse monoclonal; Cell Signaling Technology), cleaved caspase-3, caspase-3, p-ERK, ERK, p-IKKα/β, p-JNK, JNK, p-p65, and p65 (rabbit polyclonal; Cell Signaling Technology), and β-actin (mouse monoclonal; Santa Cruz Biotechnology, Dallas, CA, USA) in Tris-HCl-based buffer containing 0.2% Tween 20 (TBS-T; pH 7.5) overnight at 4 °C. The membranes were then washed and incubated with secondary monoclonal anti-mouse and polyclonal anti-rabbit antibodies (1:10,000; Santa Cruz Biotechnology) in TBS-T for 2 h. Horseradish peroxidase conjugated secondary antibody labeling was detected by enhanced chemiluminescence (ECL) using a cooled CCD camera system (ATTO Ez-Capture II; Atto Corp., Tokyo, Japan). Relative protein levels were quantified by densitometry with respect to total forms (ERK, JNK, or p65) or β-actin as the loading control.

Samples for protein analysis were prepared and analysed from fermenting, respiring and sporulating cells as published (28 (link)). Note that 25 μg of total protein extract was run on a 4–20% gradient gel (BioRad, USA) for 1 h. Proteins were transferred onto ImmobilonPSQ membranes (Millipore, France) using an electro-blotter system (TE77X; Hoefer, USA) and a modified Towbin buffer (48 mM Tris base, 40 mM glycine and 0.1% SDS) and methanol (20% vol/vol anode; 5% vol/vol cathode) for 2 h. Proteins were detected using a monoclonal anti-myc-horseradish peroxidase antibody (Life Technologies, USA) at 1:1000. The antibodies were incubated in hybridization buffer overnight. The signals were revealed using the ECL-Plus Chemiluminescence kit (GE Healthcare, USA) and the ImageQuant 350 system (GE Healthcare, USA). Band intensities were normalized and quantified using the ImageQuant TL 7.0 software and default parameters. A polyclonal antibody against Pgk1 (Invitrogen, USA) was employed as a loading control.

Cell lysates (extracted with RIPA buffer) separated on 4–12% Novex Bis-Tris SDS gels (Invitrogen, Carlsbad, CA, USA) were transferred to Immobilon-PSQ membranes (Millipore Corp. Bedford, MA, USA) with an Invitrogen western blotting system (Invitrogen. Carlsbad, CA, USA). Following blocking with 5% milk powder in 0.02% PBS-T, blots were incubated with primary antibody overnight at 4°C. Primary antibody dilutions were 1∶1000 for CDX2, fibronectin, vimentin and transgelin, 1∶2500 for β-actin and 1∶100 for SPANX-B and PAGE-2,-2B antibodies. HRP conjugated secondary antibody (Abcam, Cambridge, UK) was used at a 1∶5000 dilution and incubated at room temperature for 1 hour. Signals were detected using the ECL luminescence assay (BioRad, Hercules, CA, USA).