Fig 1: Rab32 interacts with the ER-phagy receptor Reticulon-3. A Co-immunoprecipitation of Rab32 constructs with endogenous RTN3L. MCF7 cells were transfected for 48 h with FLAG-tagged Rab32 constructs, followed by crosslinking, lysis and incubation with anti-FLAG antibodies. Immunoprecipitates were analyzed for anti-FLAG and co-immunoprecipitating endogenous long reticulon-3 (RTN3L). Densitometry of RTN3L binding normalized to respective input signals. n = 3, *p ≤ 0.05 B Co-immunoprecipitation of Rab32 constructs with endogenous Sec62, FAM134B and CCPG1. MCF7 cells were transfected for 48 h with FLAG-tagged Rab32 constructs, followed by crosslinking, lysis and incubation with anti-FLAG antibodies. Immunoprecipitates were analyzed for anti-FLAG and co-immunoprecipitating endogenous ER-phagy receptors. Densitometric FAM134B binding normalized to respective input signals. n = 3, p > 0.05. C Co-immunoprecipitation of endogenous Rab32 with HA-tagged RTN3L. MCF7 cells were transfected with HA-tagged RTN3L, followed by lysis and incubation with anti-HA antibodies. Immunoprecipitates were analyzed for anti-HA and co-immunoprecipitating endogenous Rab32. D Co-immunoprecipitation of FLAG-Rab32 constructs expressed from pcDNA3 with HA-tagged RTN3L and deletion mutants, expressed from pCEP4. HeLa cells were transfected for 48 h with FLAG-tagged Rab32 and HA-tagged RTN3L constructs, followed by crosslinking, lysis and incubation with anti-HA antibodies. Immunoprecipitates were analyzed on a 8–15% gradient gel/blot for anti-HA and co-immunoprecipitating FLAG-Rab32. The deletion mutants used were Δ1–212, Δ1–349, and Δ1–562. E Percoll fractionation of untransfected MCF7 cells for ER-phagy receptor proteins. Fractions stand for total homogenate, cytosol, microsomes, pure mitochondria and MAM (equal fractions loaded). F Immunofluorescence images of HeLa cells transfected with HA-tagged RTN3L (bottom) or empty pcDNA3 (top). Cells were incubated with antibodies against Rab32 and RTN3. Bar indicates 15 µm. Inset shows magnification of a representative area. G Immunofluorescence images of HeLa cells transfected with FLAG-tagged wild type (WT), dominant active (Q85L) or dominant negative (T39N) Rab32, HA-tagged RTN3L and control pcDNA3. Cells were incubated with Rab32 and RTN3 antibodies. Bar indicates 15 µm. Manders coefficient was calculated for Q85L and T39N. ***p ≤ 0.0001