β5-integrin primary antibody  (Assay Biotechnology)

Journal: bioRxiv

Article Title: Daam1 negatively regulates USP10 activity

doi: 10.1101/2022.02.14.480271

Figure Lengend Snippet: A) HCFs were transfected with siDaam1 or siGLO and treated with TGFβ for 3 days to increase integrin expression. siDaam1 transfection resulted in increased expression of αv-, β1-, β5-integrins. N=6. B) HCFs were transfected with siDaam1 or siGLO. After 3 days cells were lysed and subjected to Ubiquant™ ubiquitin capture ELISA. siDaam1 reduced, whereas siUSP10 increased ubiquitination of β1 and β5. N=4. (C) HCFs were transfected with siGLO or siDaam1 and treated with TGFβ for 3 days prior to processing for qPCR. The relative expression of Daam1, integrin αV, β1, and β5 is shown as compared to GAPDH. Statistical significance was calculated using an unpaired t-test Bar=200μm. N=3.

Article Snippet: Wells were then washed 4 times with TBST and then incubated in 1x Blocking Buffer in PBS with a 1:10,000 dilutions of either β1- (Assay Biotechnology; San Francisco, CA; catalogue #: R12-2927) or β5-integrin (Assay Biotechnology; San Franscisco, CA; catalogue #: F-5) primary antibody for 1 hr at room temperature.

Techniques: Transfection, Expressing, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Daam1 negatively regulates USP10 activity

doi: 10.1101/2022.02.14.480271

Figure Lengend Snippet: A) Live cell integrin recycling assay. 48 hrs post-transfection cells were blocked and treated with Ab against α5β1 or αv at 10ug/ml for 30 minutes prior to cell surface stripping for 30 sec. Cells were incubated for 90 min prior to incubation with 2° Ab-488 for 30 min. Bar=50μm. Quantification of recycled (B) Integrin α5β1 and (C) Integrin αV, N=3 with a total of 15 images analyzed for each condition. (D) Live cell FN recycling assay. HCFs were transfected as stated above. 24hrs post transfection HCFs were loaded with biotinylated-FN for 3 hours. After trypsinization (to separate cells from extracellular, non-internalized FN), HCFs were replated and imaged 48 hours post transfection. Prior to imaging, cells were incubated with streptavidin-488 to detect only recycled biotinylated-FN. Imaged by live cell confocal. Bar= 50μm. N=3. D) Quantification of extracellular FN. A total of 15 cells were analyzed for each condition from 3 independent experiments. E) Cell numbers 2 days after transfection for each condition are not significantly different. N=3. Image analysis was done using ImageJ’s Analyze Particles function. Statistical significance was calculated using an unpaired t-test +/- SEM.

Article Snippet: Wells were then washed 4 times with TBST and then incubated in 1x Blocking Buffer in PBS with a 1:10,000 dilutions of either β1- (Assay Biotechnology; San Francisco, CA; catalogue #: R12-2927) or β5-integrin (Assay Biotechnology; San Franscisco, CA; catalogue #: F-5) primary antibody for 1 hr at room temperature.

Techniques: Transfection, Stripping Membranes, Incubation, Imaging

Journal: bioRxiv

Article Title: Daam1 negatively regulates USP10 activity

doi: 10.1101/2022.02.14.480271

Figure Lengend Snippet: In response to stress-induced TGFβ1 release, both USP10 and Daam1 expression is increased. Daam1 sequesters USP10 to stress fibers, and USP10’s DUB activity on integrins is inhibited by Daam1. This may act as a level of control over USP10 activity in myofibroblasts. We further hypothesize that the known interaction of USP10 and G3BP1/2 proteins (also shown in ) positively regulates USP10’s DUB activity on integrins leading to integrin accumulation. The previously known roles of G3BP1/2 in integrin signaling support this concept.

Article Snippet: Wells were then washed 4 times with TBST and then incubated in 1x Blocking Buffer in PBS with a 1:10,000 dilutions of either β1- (Assay Biotechnology; San Francisco, CA; catalogue #: R12-2927) or β5-integrin (Assay Biotechnology; San Franscisco, CA; catalogue #: F-5) primary antibody for 1 hr at room temperature.

Techniques: Expressing, Activity Assay