Figure S2.

Proteins are ubiquitinated during pupariation in Drosophila and lysis of Vps13D-3×FLAG requires harsh conditions. (A) Top: syx17 RNAi-expressing larval intestine cells (GFP) did not have a difference in Vps13D puncta (purple) compared with control cells (non-GFP) 2 h after pupariation. Bottom: vps13d (MiMic) loss-of-function larval intestines cells (non-GFP) did not have a difference in Syx17 puncta (red) compared with control cells (green). (B) Whole-animal lysates from early third instar larval and 2 h after pupariation were lysed in harsh lysis conditions (1× Laemli buffer) and immunoblotted with antibody against ubiquitin and actin. (C) Control w¹¹¹⁸ and vps13d-3×FLAG 2-h-old prepupae were lysed in harsh lysis conditions and immunoblotted with antibody against FLAG and actin. (D) vps13d (ΔUBA) intestines expressing mitochondrial localized mito-GFP (green) throughout the intestine 2 h after pupariation were stained with a pan-ubiquitin–specific antibody (red). Scale bars in A and D are 40 μm with the exception of enlarged images, which are 5 μm.