Mouse anti β actin

The following primary antibodies were used for immunohistochemistry (IHC), immunofluorescence (IF) and western blots (WB): rabbit anti-Vangl2 (Aviva Systems Biology, San Diego, CA, OAAB15535; 1:200, IHC), rabbit anti-Vangl2 (Ramsbottom et al., 2014 (link)) (21st Century Biochemicals, Marlborough, MA; 1:2000, WB), rabbit anti-Gapdh (Millpore, Temecula, CA, ABS16; 1:30,000, WB), rabbit anti-cofilin (Cell Signaling, Beverly, MA, 5175; 1:1000, WB), rabbit anti-p-cofilin (Ser3) (Cell Signaling, 3313; 1:1000, WB), rabbit anti-β-catenin (Cell Signaling, 9562; 1:1000, WB), mouse anti-β-actin (MP Biomedicals, 08691001; 1:10,000, WB), mouse anti-E-cadherin (BD Biosciences, 610181; 1:2500, WB), rabbit anti-GM130 (Sigma-Aldrich, G7295; 1:1500, IF), rabbit anti-elastin (Biorbyt, orb13391; 1:100, WB), rabbit anti-MMP-12 (Abcam, Ab52897; 1:1000, WB and 1:200, IHC), Armenian hamster anti-Pecam1 (Abcam, Ab119341; 1:500, IF; a gift from Leo Carlin), rabbit anti-phospho-histone H3 (Ser10) (PH3; Millipore, 06-570; 1:1000, IF), rabbit anti-cleaved caspase 3 (D175) (Cell Signaling, 9661; 1:1000, IHC), goat anti-CC10 (T-18) (Santa Cruz Biotechnology, Dallas, TX, sc-9772; 1:1000, IHC), rabbit anti-proSP-C (Millipore, AB3786; 1:1000, IHC), rabbit anti-non-phospho (active) β-catenin (Cell Signaling, 8814; 1:1000, WB).

Antibodies were used at the following dilutions from the following companies: mouse anti-Cul3 (1:500, BD Biosciences, CAT#611848), rabbit anti-KCTD13 (1:1000, Atlas Antibodies, HPA043524), mouse anti-RhoA (1:500, Abnova, CAT#H00000387-M08), rabbit anti-Transgelin (1:500, Abcam, CAT#ab227566), rabbit anti-Tropomyosin-4 (1:1000, Abcam, CAT#ab181085), mouse anti-β-actin (1:1000, MP Biomedicals, CAT#691001) and mouse anti-α-tubulin (1:1000, Invitrogen, CAT# 13–8000). Secondary antibodies for Western blot were purchased from Thermo Scientific (goat anti-mouse-680, #35518 and goat anti-rabbit-800, #35571).

For western blotting, whole cell lysates were prepared in lysis buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 7.5), protease inhibitors (1 mM Na₃VO₄, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 µg/mL leupeptin, and 10 µg/mL aprotinin), and equal amounts of protein, as determined by Bradford assay, were separated by 10% SDS-PAGE and transferred to a Hybridation Nitrocellulose membrane (Millipore, Burlington, MA, USA). After blocking in 5% skimmed milk, western blots were performed using rabbit anti-AnxA6 serum [38 (link)], mouse anti-β-actin (MP Biomedicals, Illkirch-Graffenstaden, France), mouse anti-epidermal growth factor receptor kinase substrate 8 (Eps8; BD Transduction Lab, San Jose, CA, USA), rabbit anti-GFP (Abcam, Cambridge, UK), rabbit anti-Rab7 (Cell Signaling Technology, Danvers, MA, USA) and mouse anti-α-tubulin (Sigma-Aldrich, St. Louis, MO, USA). Appropriate peroxidase-conjugated secondary antibodies (BioRad Laboratories, Hercules, CA, USA) and enhanced detection (EZ-ECL; Enhanced ChemiLuminiscence, Biological Industries, Cromwell, CT, USA) for band visualization were used. The intensity of bands was quantified using ImageJ and results were normalized to actin.

For western blot analysis, the total protein concentrations were determined by a BCA assay kit (Thermo Scientific Pierce, Rockford, IL, USA). Equal amounts of protein (30 µg) were separated by gel electrophoresis on gels with the indicated percentage of polyacrylamide, and transferred onto Immobilon-P membranes (Millipore, Etten-Leur, The Netherlands). The proteins were visualized using standard protocols. Primary antibodies rabbit anti-Atg5 (ab108327), mouse anti-LC3B (ab129376), rabbit anti-Atg3 (EPR4801) (Abcam, Cambridge, UK), mouse anti-p62 (ab56416) (Abcam, Cambridge, UK) were purchased from Abcam, rabbit anti-LC3B (NB600-1384) from Novus Biologicals (Littleton, CO, USA), mouse anti-βactin from MP Biomedicals (Santa Ana, CA, USA) and rabbit anti-Atg13 from Sigma-Aldrich (St. Louis, MO, USA). Primary 4F2 mouse anti-reovirus σ3 was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biology (Iowa City, IA, USA) [20 (link)].

MDA-MB-231 cells were lysed in M-PER lysis buffer (Pierce), supplemented with protease and phosphatase inhibitor cocktail (Thermo Scientific). Protein content was measured using the Pierce BCA protein quantification Kit (Thermo Scientific). Protein samples were separated using sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (Immobilon). Membranes were blocked in 5% skimmed milk (Sigma), in tris-buffered saline (TBS) containing 0.05% Tween-20 (Sigma) and incubated overnight with primary antibodies at 4 °C and subsequently incubated with secondary antibodies for 1 h at room temperature. Primary antibodies used were mouse anti-Cyclin E1 (Abcam, ab3927, 1:500) and mouse anti-β-actin (MpBiomedicals, 69100, 1:10,000). Secondary antibodies used were horseradish peroxidase-linked anti-mouse IgG (1:2000, DAKO) and visualized using chemiluminescence (Lumi-Light, Roche Diagnostics) on a Bio-Rad bioluminescence device. Protein imaging was performed using Image Lab software (Bio-Rad). All blots derive from the same experiment and were processed in parallel.

Total protein extract was obtained by using Radioimmuno Precipitation Assay lysis buffer for five samples/group. Cell lysates containing 50 μg of total protein were then added to SDS–PAGE gels and transferred to Nitrocellulose membranes (Bio-Rad, 1704158). Membranes were blocked in 1% non-fat milk and incubated overnight at 4°C with primary antibodies, RXRα (Abcam, ab125001), and mouse anti-β-actin (MP Biomedicals, 691001). The signals were developed with an enhanced chemiluminescence solution (Bio-Rad, 1705060) and visualized on a Bio-Rad bioluminescence device. Band intensities were quantified using ImageJ and normalized to actin.