Fig. 4: TANGO6 captures cytoplasmic RPB2 and carries it into nucleus.
From: TANGO6 regulates cell proliferation via COPI vesicle-mediated RPB2 nuclear entry
a Mass spectrum results. Three candidate proteins, POLR2B (RPB2), OAT and GEMIN4, are identified from high to low scores. TANGO6 is used as a positive control. b Co-immunoprecipitation of TANGO6, RPB1 and RPB2 in HeLa cells. c The immunofluorescent staining images of RPB2 in TANGO6 WT and TANGO6 KO U251 cells. WT, wild type. KO, knock out. Scale bar, 10 μm. d The immunofluorescent staining images of RPB2 after knocking down TANGO6. Scale bar, 20 μm. e Co-immunoprecipitation of RPB2 with TANGO6 in cytoplasm (C) and nucleus (N). β-Tubulin and Histone H3 are used as internal standards of cytoplasmic and nuclear proteins respectively. f, The immunofluorescent staining images of TANGO6 and RPB2. The white and purple arrowheads indicate the co-staining and RPB2 solely signals respectively. Scale bar, 10 μm. g Western blot analysis of TANGO6, RPB1/2, COP vesicle (COPA, Sec31A and ERGIC53), cis-Golgi (GM130) and ER (Calnexin) in each sucrose gradient fraction (from 10% to 37%). h Co-immunoprecipitation of HA-TANGO6, RPB2 and COPA (COPI vesicle marker) after extraction of cis-Golgi and trans-Golgi components. i The immunofluorescent staining images of RPB2 after knocking down COPA. Scale bar, 20 μm. j Co-immunoprecipitation of HA-TANGO6 and RPB2 when knocking down COPA. The diagram of GFP fused N- (1–146 aa) terminal (GFP-N), Cytoplasmic (161–472 aa, GFP-cyto) and C- (487–1094 aa) terminal (GFP-C) fragments (k) and Co-immunoprecipitation of TANGO6 different fragments with RPB2 (l). m Co-immunoprecipitation of HA-TANGO6 with RPB2 in different phases of cell cycle. n The immunofluorescent staining images of TANGO6, RPB2 and Calnexin. The down panels are the amplified images of boxed region. The white arrowheads indicate the merged signals of TANGO6, RPB2 with Calnexin. Scale bar, 10 μm. o The immunofluorescent staining images of RPB2 when knocking down KPNB1. Scale bar, 20 μm. p The diagram shows that COPI-docked TANGO6 captures RPB2 via its cytoplasmic N- and C- terminal fragments and directs it nuclear entry via the assistance of ER and KPNB1. The models were created with BioRender.com. Source data are provided as a Source Data file.
