Fig 1: Biochemical characterization of CPSF6, NUP153 and SEC24C interactions with HIV-1 cores and CA tubes.Representative immunoblots showing CA nanotube mediated co-pelleting of endogenous CPSF6 (a), NUP153 (b) and SEC24C (c) from MT4 cell lysates. The proteins of interest were visualized by antibodies ab175237(Abcam) against CPSF6, NB100-93329 (Novus) against NUP153, ab122633 (Abcam) against SEC24C. Lane 1: cell lysate. Lane 2: supernatant or unbound fraction after pelleting in the absence of CA tubes. Lane 3: supernatant or unbound fraction after co-pelleting with pre-formed CA tubes. Lane 4: pelleted or bound fraction in the absence of CA tubes. Lane 5: pelleted or bound fraction in the presence of CA tubes. The experiments were repeated 3 times independently with similar results. Quantitation of GST-mediated affinity pull-down of native HIV-1 cores bound to indicated concentrations of GST-CPSF6261-358(LCR-FG-LCR) vs GST-CPSF6(FG)/nonLCR (d), GST-NUP1531306-1450(LCR-FG-LCR) vs GST-NUP153(FG)/nonLCR (e); and GST-SEC24C196-314(LCR-FG-LCR) vs GST-SEC24C(FG)/nonLCR (f). The results were analyzed by Origin 2019 (v.9.6) software to determine binding Kd values. Each data point represents mean values + /− SD from three independent experiments. Source data are provided as a Source Data file. g Quantitation of GST-mediated affinity pull-down of isolated native HIV-1 cores vs crosslinked CA hexamers with 2 μM GST-CPSF6261-358(LCR-FG-LCR) (left), GST-NUP1531306-1450(LCR-FG-LCR) (middle) and GST-SEC24C196-314(LCR-FG-LCR) (right). Mean values + /− SD from three independent experiments are shown. Source data are provided as a Source Data file. h Representative immunoblots of three independent experiments showing co-pelleting of GST-CPSF6261-358(LCR-FG-LCR) (top), GST-NUP1531306-1450(LCR-FG-LCR) (middle) and GST-SEC24C196-314(LCR-FG-LCR) (bottom) with pre-formed CA nanotubes. The experiments were repeated 3 times independently with similar results.
Fig 2: ERES disintegrate in TEPCR2 deficient cells.a Immunoblot analysis of TECPR2 WT and MUT cells as well as TECPR2 MUT cells re-expressing TECPR2 WT and L440Rfs. PCNA served as loading control. b TECPR2 WT and MUT cells were fixed and immunolabeled with anti-SEC13 or -SEC24C. Nuclei were stained with DRAQ5. Statistical two-sided t-test analysis of normalized SEC24C and SEC13 spots (n = 4 independent experiments). Error bars represent mean ± SEM, p value = 0.00003 and 0.01064 for SEC24C and p value <0.00001 and =0.00846 for SEC13. Scale bars 10 µm. c Immunoblotting of fibroblasts expressing WT or L440Rfs*19-mutant TECPR2. d Fibroblasts were fixed and immunolabeled with anti-SEC13 or -SEC24C. Nuclei were stained with DRAQ5. Statistical two-sided t-test analysis of normalized SEC24C and SEC13 spots (n = 4 independent experiments). Error bars represent mean ± SEM, p value = 0.00001 for SEC24C and p value = 0.00021 for SEC13. Scale bars 10 µm. e TECPR2 WT and MUT cells were fixed and immunolabeled with anti-SEC13 or -SEC24C together with an anti-SEC31A antibody. Insets show magnification of boxed areas. Scale bars 10 µm. Quantification of area overlap SEC13/SEC24C with SEC31A (normalized to WT, two-sided t-test analysis, n = 4 independent experiments, error bars represent mean ± SEM, p value <0.00001 for SEC13 and p value = 0.00699 for SEC24C.). f TECPR2 WT and MUT cells expressing APEX2-SEC13 were subjected to biotinylation followed by lysis and streptavidin pulldown (S-PD). TL, total lysates. TECPR2 WT and MUT cells expressing APEX2-SEC13 (g) or APEX2-SEC16A (h) were subjected to biotinylation followed by lysis and streptavidin pulldown (S-PD). TL total lysates. i Homogenates from TECPR2 WT cells were left untreated or incubated with proteinase K, RapiGest or both. BiP and Tubulin served as controls. Source data are provided as a Source Data file.
Fig 3: Analysis of the colocalization between SEC31A and SURF4 or SEC24C.A–C, the localizations of SEC31A and SURF4 were analyzed in HeLa cells (The scale bar represents 10 μm). Magnified views of the indicated areas in panel C are shown in panels C’–C’’’’ (The scale bar represents 2 μm). D, diagram depicting permeabilized cell assay. E–P, HeLa cells were permeabilized by digitonin and incubated with the indicated reagents. After incubation, the localizations of the indicated proteins were analyzed by immunofluorescence (The scale bar represents 10 μm). Magnification, 63×. Magnified views of the indicated areas in panels J and P are shown in panels J’, J’’, P’ and P’’ (The scale bar represents 2 μm). Q, quantification of the colocalization between SEC31A and SURF4 or SEC24C (n = 3, mean ± S.D., over 25 cells from five random imaging fields were quantified in each experimental group). ∗∗p < 0.01. R, quantifications of the total above-threshold fluorescent level of the signal detected by antibodies against SURF4, SEC31A, or SEC24C in each cell (mean ± SD; five random fields per group are quantified; >20 cells were quantified in each field). One representative experiment from three biological repeats is shown. ∗p < 0.05; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. SURF4, surfeit locus protein 4.
Fig 4: Delayed and modular SG recruitment of late dynamic proteins.a Immunoblots of the total input and the SG fraction samples collected at indicated HS and recovery time points for the detection of IARS, QARS, KARS, EPRS, and RARS; CAPRIN1 is a SG constituent control in the SG fraction; representative blots from 3 biological replicates are shown. b, d, f Confocal micrographs of U2OS cells fixed at the indicated time points during HS and recovery; immunofluorescence of G3BP1 and eIF3A (b), VCP (d), or SEC24C (f) are shown, SGs were indicated by G3BP1, presented in red, other proteins were presented in green; arrows (b) or insets (f) highlight colocalization of two proteins, scale bars, 20 μm; top of (e), z-stack projections, scale bars, 10 μm; middle of (e), magnified orthogonal sectioning view of regions in boxes, scale bars, 4 μm; bottom of (e): line profiles of G3BP1 and VCP fluorescence intensity along the white solid lines; representative images from 3 biological replicates are shown. c, e, g Enrichment of eIF3A (c), VCP (e), and SEC24C (g) fluorescence intensity in G3BP1-positive granules over that in the whole cell, calculated per cell; error bars indicate SEM; the fitted trend of late proteins based on proteomic profiling results (Fig. 1f) is shown in blue as a reference; n = 102 (c), 100 (e, g) cells for every time point, from 3 biological replicates. Source data are provided as a Source Data file.
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