ab24693 staining Integrin beta 1 in wild-type HAP1 cells (top panel) and Integrin beta 1 knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab24693 at 5μg/ml concentration and ab202272 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Mouse IgG (Alexa Fluor^® 488) (ab150117) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

Overlay histogram showing HAP1 wildtype (green line) and HAP1-ITGB1 knockout cells (red line) stained with ab24693. Live HAP1 wildtype and HAP1-ITGB1 knockout cells were incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab24693, 0.1µg/0.5x10⁶ cells) for 30 min at 22°C. A mouse IgG1 isotype control antibody  (ab170190) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-ITGB1 knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity). Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.

Cells were exposed to inhibitors for 60 min and then fixed and stained for the presence of β1-integrin (greyscale and red in the merge) and the plasma membrane (green in the merge). LSCM images were acquired at the cell-substrate interface. (A) Control (scale bar = 20 µm and applies to all), (B) Cyt-D, (C) nocodazole, (D) Y-27632A, (E) ML-7 and (F) blebbistatin treated cells. In all cases, integrin-ß1 is well distributed over the cell contact area. However, after 60 min of CytD treatment a significant decrease in integrin-ß1 was observed, correlating to a significant decrease in cell adhesion strength (Fig. 5).

Cells were first fixed by parafromaldehyde and subsequently extracted by methanol. ab24693 and Alexa Fluor^® 546 rabbit anti-mouse immunoglobin were used as primary and secondary antibodies, respectively. Cells were also stained with wheat germ agglutinin coupled to Oregon Green 488 to reveal the cell membrane.

Overlay histogram showing MCF7 cells stained with ab24693 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab24693, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was a goat anti-mouse DyLight^® 488 (IgG; H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in MCF7 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

ICC/IF image of ab24693 stained HeLa cells. The cells were formaldehyde fixed, permeabilization with 0.5% TritonX/PBS and then blockedwith 5% BSA for 30 minutes at 21°C. The cells were then incubated with the antibody (ab24693, 1/200) for 1 hour at 21°C. The secondary antibody (green) was Alexa Fluor^® 488 goat anti-mouse IgG (H+L) used at a 1/500 dilution. DAPI was used to stain the cell nuclei (blue) and Phalloidin-TRITC (red) the actin cytoskeleton.

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Higher intensity for integrin β₁ staining can be observed in the low shear sample compared to no shear.

a) Control (no shear) sample, b) After 24 hours of low shear stress exposure.

Control cells and cells exposed to low shear for 24-hr were fixed with 4% paraformaldehyde. Samples were permeabilized with 0.1% Triton and nonspecific binding was blocked with 1% BSA/10% goat serum. Samples were incubated with ab24693 followed by Alexa Fluor^® 488 conjugated secondary antibody (ab150113).