Fig 1: AKIP1 knockdown inhibits the invasion and migration of ccRCC cells. (A) The invasion (magnification, x100) and (B) migration of Caki-1 cells transfected with siRNA-AKIP1 were determined by Transwell and wound healing assays (magnification, x200). (C) The expression of metastasis-related proteins in Caki-1 cells transfected with siRNA-AKIP1 was detected by western blotting. ***P<0.001 vs. control group; ###P<0.001 vs. siRNA-NC group. ccRCC, clear cell renal cell carcinoma; AKIP1, a-kinase interaction protein 1; siRNA, small interfering RNA; NC, negative control.
Fig 2: EBOV VP35 activates PKA via AKIP1 association.a Lysates of HEK293 cells cotransfected with the indicated plasmids were incubated with/without recombinant His-VP35 for 2 h and then subjected to immunoprecipitation and immunoblotting analysis. b Wild-type (WT) and AKIP1 knockout (KO) HepG2 cells transfected with GFP-VP35 or GFP were treated with FSK (25 µM) for 45 min and were subjected to anti-PRKACA immunostaining (red). Arrow: cells expressing GFP-VP35. c Lysates of HepG2 cells transfected with Flag-vector, Flag-VP35, or Flag-VP35 mutant plasmids were analyzed by immunoblotting using a PKA substrate phosphorylation antibody. d Lysates of WT and AKIP1-/- HepG2 cells infected with Ad-VP35 or Ad-null (MOI = 10) were subjected to PKA activity assays. Differences between the two groups were evaluated using a two-sided unpaired Student’s t-test. Data were presented as mean ± s.e.m. ns not significant; **P < 0.01. e Concentrations of cAMP were detected in the lysates of WT and AKIP1-/- HepG2 cells infected with live EBOV (MOI = 10). Differences between the two groups were evaluated using a two-sided unpaired Student’s t-test. Data from three independent experiments were analyzed, are presented as the means ± s.e.m. (**P < 0.01; ***P < 0.001).
Fig 3: AKIP1 participates in the angiogenesis in ccRCC by binding to Rac1. (A) The angiogenesis in CM from Caki-1 cells transfected with siRNA-AKIP1 and Ov-Rac1 was detected by HUVEC tube-formation assay. The (B) mRNA and (C) protein expression of angiogenesis-related proteins in CM from Caki-1 cells transfected with siRNA-AKIP1 and Ov-Rac1 were detected by reverse transcription-quantitative PCR and western blotting. ***P<0.001 vs. control (CM)+ HUVEC group; ##P<0.01 and ###P<0.001 vs. siRNA-AKIP1 (CM)+HUVEC group; ++P<0.01 and +++P<0.001 vs. siRNA-AKIP1+Ov-NC (CM)+HUVEC group. CM, culture media; ccRCC, clear cell renal cell carcinoma; AKIP1, a-kinase interaction protein 1; siRNA, small interfering RNA; NC, negative control; Ov, overexpression.
Fig 4: AKIP1 knockdown inhibits the proliferation of ccRCC cells. (A) mRNA (B) and protein expression of AKIP1 in Caki-1 cells transfected with siRNA-AKIP1-1 and siRNA-AKIP1-2 were detected by reverse transcription-quantitative PCR and western blotting. (C) The viability of Caki-1 cells transfected with siRNA-AKIP1 was analyzed by Cell Counting kit-8 assay. (D) The expression of proliferation-related proteins in Caki-1 cells transfected with siRNA-AKIP1 was detected by western blotting. (E) The proliferation of Caki-1 cells transfected with siRNA-AKIP1 was analyzed by 5-Ethynyl-2'-deoxyuridine staining (magnification, x200). **P<0.01 and ***P<0.001 vs. control group; ##P<0.01 and ###P<0.001 vs. siRNA-NC group. ccRCC, clear cell renal cell carcinoma; AKIP1, a-kinase interaction protein 1; siRNA, small interfering RNA; NC, negative control.
Fig 5: EBOV VP35 interacts with AKIP1 in the viral inclusion bodies of HepG2 cells infected with EBOV or trVLPs.a HepG2 cells infected with Zaire EBOV (strain Mayinga) (MOI = 10) for 72 h were analyzed by immunostaining with anti-VP35 (green) and anti-AKIP1 (red) antibodies (upper panel) or anti-VP35 antibody only (lower panel). b HepG2 cells infected with live EBOV (MOI = 10) for 72 h or transfected with the EBOV minigenome (p0) for 48 h were subjected to an in situ PLA assay with anti-VP35 and anti-AKIP1 antibodies (red), and immunostaining with an anti-NP antibody (green). Arrows: VP35-AKIP1 complexes in the viral inclusion bodies of EBOV (upper panel) or trVLPs (lower panel). At least three independent repeats were performed in all of the experiments.
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