antibody against phospho paxillin  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Integrin β1–Talin1 at focal adhesions underpin uncontrolled endothelial cell enlargement in live cerebral cavernous malformation vasculature

doi: 10.1101/2025.11.25.688491

Figure Lengend Snippet: (a) Western blot of WT and CCM1 LOF ECs demonstrated a loss of CCM1 (KRIT1) protein expression. (b) Western blot of WT and CCM1 LOF HUVECs demonstrated an increase in Integrin β1 protein expression. (c) Quantification of Integrin β1 protein expression, corrected to loading control and normalized to WT HUVECs. 6 biological replicates, unpaired two-tailed Student’s t-test. (d) Maximum intensity projection of confluent monolayers of WT and CCM1 LOF ECs with immunofluorescent staining phospho-Paxillin (Tyr118, magenta), Vinculin (cyan), VE-cadherin (yellow), and nuclei (DAPI, grey). Heatmap of Vinculin expression, with magenta arrowhead indicating Vinculin at AJs and cyan arrowhead indicating Vinculin at FAs (scale bar = 20 μm). (e) Quantification of the number of FAs per EC and (f) average FA area comparing WT ECs to CCM1 LOF ECs, based on pPaxillin expression. (g) Quantification of the ratio of Vinculin over VE-cadherin, indicating cadherin AJs under mechanical tension (h) Quantification of the ratio of Vinculin over pPaxillin, indicating FAs under mechanical tension. 3 biological replicates, 5-8 images per replicate, with each data point representing an average of 4-24 cells per image. Unpaired two-tailed Student’s t-test (F, G) and unpaired two-tailed Student’s t-test with Welch’s correction (E, H). Data represented using violin plots or bar plots, small circles indicate individual data points for each replicate (color matched to replicate). *p=0.0332, **p=0.0021, ***p=0.0002, ****p<0.0001.

Article Snippet: The following antibodies were used: rabbit α-KRIT1 (CCM1) (WB 1:1000; Abcam, ab196025), rabbit α- GAPDH (WB 1:5000; Cell Signalling, 2118), mouse α-VE-cadherin conjugated to Alexa Fluoro 647 (IF 1:500, Becton Dickinson, 561567), rabbit α-phosphorylated Paxillin (Y118) (IF 1:250; Cell Signalling, 69363), mouse α-Vinculin (1:250; Sigma Aldrich, V9131), rabbit α-KLF4 (IF 1:100; Cell Signalling, 4038), rat α-mouse CD29 (9EG7) (IF 1:200; Becton Dickson, 550531), rabbit α-ITGB1 (WB 1:1000; Sapphire Bioscience, STJ93731).

Techniques: Western Blot, Expressing, Control, Two Tailed Test, Staining