Figure 5

RACK1 depletion modulates mRNA and protein levels of TIMAP and affects the membrane localization of TIMAP. (A) HPAEC grown on 6-well plate was transfected with small interfering RNA (siRNA) as described in Materials and Methods. Total RNA was isolated from non transfected (ctr), non silencing RNA, or RACK1 specific siRNA (siGBN2L1) transfected cells and analyzed by RT-PCR using specific primer pairs for RACK1, PP2A B (irrelevant control) or TIMAP. (B) Western blot analysis of non transfected (ctr), non silencing RNA, or siGBN2L1 transfected cells using RACK1, TIMAP and actin specific antibodies. Actin was tested as loading control. The amount of RACK1 or TIMAP signal was expressed as ratio of RACK1/TIMAP:β-actin signal density. The error bars correspond to SE from 3 independent transfections. Statistical analysis was done with Student’s t-test. Significant changes are indicated by asterisks; ** (P < 0.01), or *** (P < 0.001). (C) Immunofluorescence staining of confluent non transfected (ctr) (a-c), non silencing RNA (d-f) or siGBN2L1 treated (g-i) HPAEC using anti-TIMAP (a,d,g: green) and anti-RACK1 (b,e,h: red) primary antibodies is presented. Scale bars: 100 μm. (D) Membrane fractions of non transfected (ctr), non silencing RNA or siGBN2L1 transfected HPAEC were isolated as described in Materials and Methods. Total cell lysate (CL) was also loaded as control. The fractions were analyzed with anti-TIMAP and anti-CD31 antibodies. Shown are representative data of at least 3 independent experiments.