scotin knockout cell generation  (Santa Cruz Biotechnology)

Journal: Developmental cell

Article Title: Intrinsically disordered region-mediated condensation of IFN-inducible SCOTIN/SHISA-5 inhibits ER-to-Golgi vesicle transport.

doi: 10.1016/j.devcel.2023.08.030

Figure Lengend Snippet: Figure 1. SCOTIN inhibits transport from ER to Golgi (A) Schematic illustration of proximal profiling of SCOTIN with BioID. (B) Gene ontology (GO) analysis of proteins identified in the proximal profiling. (C) Schematic illustration of the RUSH reporter system. (D) Cell surface trafficking of E-cadherin-SBP-EGFP was assessed after expression of indicated constructs followed by biotin treatment. (E) Live confocal images of HeLa cells transfected with the E-cadherin-SBP-EGFP with empty or SCOTIN-MYC plasmids during the first 20 min after biotin treatment. (F) (Top) Confocal images of HeLa cells expressing E-cadherin-SBP-EGFP with empty, or SCOTIN-MYC with or without biotin (15 min) followed by immuno- staining with MYC and giantin. (Bottom) The Mander’s coefficient of EGFP overlapping giantin for each cell (n = 6–12). (G) (Top) Confocal images of HeLa cells expressing SBP-EGFP-GPI with empty, or SCOTIN-MYC with or without biotin (7 min) followed by immunostaining with MYC and giantin. (Bottom) The Mander’s coefficient of EGFP overlapping GM130 for each cell (n = 15–17). (H) The trafficking of VSV-Gts045 from ER to Golgi was observed after temperature shift from 40C to 32C for 10 min. Confocal images of HeLa cells after im- munostaining of VSV-G and giantin (top), and Mander’s coefficient of VSV-G overlapping giantin (bottom) were shown (n = 6–8). All graphs are represented as mean ± SEM. All scale bars, 10mm. ***p < 0.001

Article Snippet: SCOTIN KO cell generation with CRISPR/Cas9-mediated genome editing To generate a SCOTIN KO HeLa cell line, a gRNA/Cas9 expression plasmid and a donor plasmid were purchased from Santa Cruz Biotechnology (Table S1).

Techniques: Expressing, Construct, Transfection, Immunostaining

Journal: Developmental cell

Article Title: Intrinsically disordered region-mediated condensation of IFN-inducible SCOTIN/SHISA-5 inhibits ER-to-Golgi vesicle transport.

doi: 10.1016/j.devcel.2023.08.030

Figure Lengend Snippet: Figure 3. Endogenous SCOTIN hijacks Sec31 and interferes ER-to-Golgi transport upon IFN-g stimulation (A) The absence of SCOTIN in the generated SCOTIN KO or KO#2 cells was confirmed by immunoblotting. (B and C) ER-to-Golgi trafficking of the RUSH reporter was compared in WT and SCOTIN KO cells. Cargos are E-cadherin (n = 21–23) in (B) and GPI (n = 19–21) in (C).

Article Snippet: SCOTIN KO cell generation with CRISPR/Cas9-mediated genome editing To generate a SCOTIN KO HeLa cell line, a gRNA/Cas9 expression plasmid and a donor plasmid were purchased from Santa Cruz Biotechnology (Table S1).

Techniques: Generated, Western Blot

Journal: Developmental cell

Article Title: Intrinsically disordered region-mediated condensation of IFN-inducible SCOTIN/SHISA-5 inhibits ER-to-Golgi vesicle transport.

doi: 10.1016/j.devcel.2023.08.030

Figure Lengend Snippet: Figure 5. SCOTIN condensates are formed with the tubular ER (A–C) Confocal immunofluorescence images of SCOTIN-MYC relative to RTN4 (A), Sec61b (B), or CLIMP63 (C). Scale bars, 10 mm. (D) Airyscan immunofluorescence images of SCOTIN-MYC and RTN4. Representative features are illustrated in the right panel. Scale bars, 10 mm. (E) CLEM images of HeLa cells expressing SCOTIN-mDsRed. The white arrows indicate SCOTIN puncta near the ER membrane. The arrowheads, MT, and v indicate ER, mitochondria, and intracellular vesicles, respectively. Scale bars, 1 mm. (F) CLEM image of HeLa cells expressing SCOTIN(D150–177)-mDsRed. Scale bars, 1 mm. (G) Confocal immunofluorescence images of SCOTIN(D150–177)-mDsRed relative to RTN4. Scale bars, 10 mm.

Article Snippet: SCOTIN KO cell generation with CRISPR/Cas9-mediated genome editing To generate a SCOTIN KO HeLa cell line, a gRNA/Cas9 expression plasmid and a donor plasmid were purchased from Santa Cruz Biotechnology (Table S1).

Techniques: Expressing, Membrane