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Figure 4 | Cell Communication and Signaling

Figure 4

From: RACK1 is involved in endothelial barrier regulation via its two novel interacting partners

Figure 4

GSK3β inhibitor results in loss of membrane localized TIMAP. (A) Immunofluorescence staining of confluent HPAEC without (a-c) (CTR), or with various treatments as follows: 50 μM forskolin (FRSK) for 30 min (d-f); 20 μM GSK3β inhibitor, AR-A014418, (GSK3 inh) for 4 hrs (g-i); or 20 μM GSK3β inhibitor for 4 hrs followed by 50 μM forskolin for 30 min (j-l) using anti-TIMAP (a,d,g,j: green) and anti-RACK1 (b,e,h,k: red) primary antibodies was performed. Representative data of at least three independent experiments are shown. Scale bars: 100 μm. (B) Subcellular fractionations of HPAEC cells after the same set of treatments as listed in panel (A) were made as described in Materials and Methods. The fractions were analyzed with anti-TIMAP, anti-CD31 as membrane, anti-lamin A/C as nuclear and anti-β-tubulin (not shown) as cytoplasmic marker antibody. Shown are representative data of at least 3 independent experiments. Quantitative analysis of TIMAP signals is also shown. CD31 or lamin A/C bands were used for protein level normalization. The results are presented as means ± SE from 3 independent experiments. Statistical analysis was done with Student’s t-test. Significant changes are indicated by asterisks; * (P < 0.05), ** (P < 0.01), or *** (P < 0.001). (C) TIMAP was immunoprecipitated from HPAEC after the same set of treatments as listed in panel (A). Total cell lysates and the IP complexes were probed for TIMAP and RACK1. Additional bands in IP samples correspond to IgG.

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