Mouse anti β gal

Immunostaining of imaginal discs were performed with previous protocols^{48 (link)}. In brief, third-instar larvae were dissected in PBS and fixed in freshly made 4% formaldehyde in phosphate buffered solution (PBS) at room temperature for 20 min, then washed three times with PBT (PBS plus 0.1% Triton X-100). Larvae were incubated overnight with needed primary antibodies in PBT at 4 °C, then washed with PBT for three times and incubated with corresponding fluorophore-conjugated secondary antibody 2 h at room temperature. After washing for three times in PBT, discs were dissected and mounted in 40% glycerol. For immunostaining of S2 cells, cells were transfected with indicated constructs and stained with previous protocols^{62 (link)}. Images were captured with Zeiss confocal microscope. Antibodies used in this study were as follows: rat anti-Ci (1:50; DSHB); mouse anti-β Gal (1:500; Santa Cruz); goat anti-CycE (1:100; Santa Cruz); mouse anti-FgM2 (1:1000; Sigma); mouse anti-BrdU (1:10; DSHB); rabbit anti-Yki (1:1000), mouse anti-Myc (1:200; Santa Cruz), and DAPI (1:1000; Santa Cruz). Rabbit anti-DIAP1 antibody (1:100) was from Dr. Shigeo Hayashi lab. Mouse anti-Usp7 (1:100) was generated using aa1-234 as the antigen. Secondary antibodies used in this study were bought from Jackson ImmunoResearch, and were diluted at 1:500.

Immunostaining of wing and eye disks was carried out according to our previous protocols [54 (link)]. Briefly, third-instar larvae were dissected in PBS and fixed in freshly made 4% formaldehyde in PBS at room temperature for 20 min, then washed three times with PBT (PBS supplemented with 0.1% Triton X-100). Larvae were incubated overnight with primary antibodies in PBT at 4 °C, then washed with PBT for three times and incubated with fluorophore-conjugated secondary antibodies for 2 h at room temperature. After washed for three times in PBT, disks were separated and mounted with 40% glycerol. Images were captured with Zeiss confocal microscope. Primary antibodies used in this study were shown as follows: rabbit anti-PH3 (1:100, Abcam); rat anti-Ci (1:50, DSHB); rabbit anti-Cas3 (1:100, ABclonal); mouse anti-β Gal (1:500, Santa Cruz); mouse anti-HA (1:200, Santa Cruz); mouse anti-Myc (1:200, Santa Cruz); rabbit anti-DrICE (1:100, CST) and rabbit anti-DIAP1 (1:100) [55 (link)]. All secondary antibodies used in this study were bought from Jackson ImmunoResearch, and were diluted at 1:500.