Journal: Nature Communications

Article Title: Spatial proteomics reveals secretory pathway disturbances caused by neuropathy-associated TECPR2

doi: 10.1038/s41467-023-36553-6

Figure Lengend Snippet: a Electron micrographs of APEX2-TECPR2 WT or L440Rfs expressing cells. Before embedding and ultrathin sectioning, fixed samples were incubated with DAB and H 2 O 2 . Insets show magnification of representative nuclear and vesicular areas. Scale bars 200 nm. b TECPR2 WT and MUT cells were subjected to homogenization and differential centrifugation. Cytosolic (CYTO) and total membrane (TM) fractions were analyzed by immunoblotting. GM130 and tubulin served as loading controls. TECPR2 WT and MUT cells differentially treated with an ATG7 inhibitor ( c ) or ATG8-HEXA KO HeLa cells ( d ) were subjected to homogenization and differential centrifugation. Total lysate (TL), cytosolic (CYTO) and total membrane (TM) fractions were analyzed by immunoblotting. GM130, LC3B, GABARAP and tubulin served as controls. e Schematics of two-step differential centrifugation, resulting in total lysates (TL), nuclear debris (N), cytosol (C), total membranes (TM), light sucrose membranes (L), a sucrose membrane pellet (P) and six different OptiPrep membrane fractions (1–6). f TECPR2 WT cells were subjected to homogenization followed by two-step differential centrifugation and immunoblotting. g HA-TECPR2 WT or L440Rfs were immune-isolated from homogenates and selected association partners detected by immunoblotting. DTNBP1 served as loading control. HA-IP, immune-isolated membranes. h Working model of TECPR2’s field of operations at the ER-Golgi interface and protein sorting defects caused by truncated TECPR2. Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used:ADAMTS1 (Abcam, #ab39194), APEX2 (IgG2A) (custom made by Regina Feederle, HZM München), ATP1A1 (Abcam, #ab7671), B4GALT3 (Proteintech, #11041-1-AP), BAG2 (Biomol, #A304-751A), beta-ACTIN (Sigma,#A1978), BIOTIN (Pierce, #31852), BiP (Cell Signaling, #3177), CALNEXIN (Abcam, #ab22595), Calreticulin (santa cruz, #sc-6467), CD63 (abcam, #ab59479), CLPTM1L (Sigma, #HPA014791), c-myc (Bethyl, #A190-104A), CRLF1 (Novus, #NBP1-85606), DTNBP1 (Bethyl, #A303-360A), EDIL3 (Abcam, #ab190692), ERGIC1 (Proteintech, #16108-1-AP), ERGIC53 (Santa Cruz, #sc398777), EXTL2 (Abcam, #ab168391), Flag M2 (Cell Signaling, #2368), GABARAP(Abcam,#109364), Giantin (Biolegend, #924302), GM130 (Abcam, #ab52649), GOLIM4 (Abcam, #ab28049), HA.11 Clone 16B2 (Covance/Biolegend, #MMS-101P/#901501), HS6ST2 (Abcam, # ab122220), LAMP1 (IF 1:100, Abcam/DSHB, #ab24170/H4A3), LAMP2 (IF 1:100, Abcam, #ab25631), LAMTOR1 (Cell Signaling, #8975S), LAMTOR2 (Cell Signaling, #8145); LAMTOR3 (Cell Signaling, #8168), LC3B (Cell Signaling, #2775S), LGALS3BP (Proteintech, #10281-1-AP), LRP1 (Abcam, #ab92544), M6PR (Abcam, #ab2733), MTOR (Cell Signaling, #2983), myc 9E10 (custom made by Regina Feederle, HZM), NCAM1 (WB: 1:500, IF 1:100, Merck, #AB5032), NEK9 (Abcam, #ab138488), NID1 (Invitrogen, #PA5-99666), NPC1 (WB: 1:500, Abcam, #ab134113), PCNA (Santa Cruz, #sc-7907), PLXDC2 (Novus, #NBP1-76858), PLXNA1 (R&D, #AF4309), PLXNA2 (Abcam, #ab39357), RAB5C (Sigma,#HPA003426), SEC12 (Novus, #NBP1-87056), SEC13 (Novus, #AF9055-100), SEC24C (Abcam, #ab122633), SEC24D (Cell Signaling, #14687), SEC31A (BD, #612351), SLC38A9 (Abcam, #ab81687), SPG20 (Proteintech, #13791-1-AP), TECPR2 (Christian Behrends, custom made), TNC (Abcam, #ab108930), TOLLIP (Abcam, #ab187198), TOMM40 (Abcam, #ab185543), TRAPPC11 (Sigma, #HPA045427), TRAPPC8 (Sigma, #HPA041107), TRAPPC9 (Proteintech, #16014-1-AP), TSG101 (Abcam, #ab30871), TUBULIN (Abcam, #ab7291), VAPA (Sigma, # HPA009174), VAPB (Sigma, # HPA013144), VCP (Bethyl, #A300-588A), VPS11 (Abcam, #ab125083).

Techniques: Expressing, Incubation, Homogenization, Centrifugation, Membrane, Western Blot, Isolation, Control