Figure 5.

The TECPR domain of TECPR2 associates with autophagosomes and lysosomes. (A) Schematic presentation of TECPR2 domains fused to mCherry. (B) HeLa cells were transfected for mCherry-TECPR2 expression using JetPrime reagent for 24 h. Cells were fixed with methanol, immunostained for LC3B and LAMP1 and analyzed by confocal microscopy. Scale bar: 20 μm and 2 μm for zoomed images. (C) Proteins extracted from cells transfected as (B) were floated on a sucrose gradient as described in Materials and Methods, collected fraction proteins were immunoblotted for mCherry, LC3B and VAMP8. (D) HeLa cells were transfected with siRNA targeting VAMP8 and/or STX17, or a non-targeting (siNT) control, using DharmaFECT1 transfection reagent. After 24 h cells were transfected for mCherry-TECPR or mCherry-TECPR[ΔLIR] expression using JetPrime reagent for additional 24 h. Cells were fixed with methanol, immunostained for LC3B and LAMP1 and examined by confocal microscopy. Scale bar: 2 μm. Large magnifications of stained cells are presented. Colocalization of structures positive for mCherry-TECPR2 or mCherry-TECPR2[ΔLIR] with LC3B, LAMP1 or both was quantified manually