Anti β4 clone 7

Y-27632 and 4’,6-diamidino-2-phenylindol (DAPI) were purchased from Wako Pure Chemical Industries (Osaka, Japan) and Polysciences (Warrington, PA), respectively. Matrigel (growth factor-reduced) was purchased from Corning Incorporated (Corning, NY). Rabbit polyclonal antibodies against human Solo were prepared as previously described [14 (link)]. Other antibodies were purchased as follows: anti-FLAG (M2; Sigma-Aldrich, St. Louis, MO), anti-GFP (A6455; Life Technologies, Camarillo, CA), anti-β-actin (AC-15; Sigma-Aldrich), anti-K18 (DA-7; BioLegend, San Diego, CA), anti-GM130 (CSB-PA600856ESR1HU; Flarebio Biotech LLC, College Park, MD), anti-β4 (58XB4; BioLegend) for immunofluorescence, and anti-β4 (Clone 7; BD Biosciences, Franklin Lakes, NJ) for immunoblotting. Secondary antibodies, anti-mouse IgG-Alexa 488, anti-mouse IgG-Alexa 568, anti-rabbit IgG-Alexa 568, and anti-rat IgG-FITC, were purchased from Thermo Fisher Scientific (Waltham, MA).

To visualize and quantify the integrin proteins, RT112 cell lysates were applied to a 7% polyacrylamide gel and electrophoresed for 90 min at 100 V. The proteins were then transferred to nitrocellulose membranes (1 h, 100 V). After this, membranes were blocked with non-fat dry milk for 1 h and incubated overnight with monoclonal antibodies directed against the following integrin proteins: anti-α2 (clone 2, 1:250), anti- α3 (1:500; both: Merck Millipore, Darmstadt, Germany), anti- α5 (clone 1, 1:5000), anti- αV (clone 21/CD51, 1:250), anti-β1 (clone 18, 1:2500), anti- β3 (clone 1, 1:2500), or anti- β4 (clone 7, 1:250; all: BD Biosciences) and anti- α6 (dilution 1:1000, Cell Signaling, Frankfurt, Germany). HRP-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-rabbit IgG (both: Cell Signaling) served as the secondary antibodies. Membranes were briefly incubated with chemiluminescence (ECL) detection reagent (Amersham/GE Healthcare, München, Germany) to visualize the proteins and then analyzed using the Fusion FX7 system (Peqlab, Erlangen, Germany). β-actin (Cell Signaling) served as the internal control. GIMP 2.8 software was used to analyze the pixel density of the protein bands and to calculate the ratio of protein intensity/β-actin intensity.