α dperk

All immunostainings were carried out following protocols described in^{20 (link)}. Antibodies used in this study: α-Discs large (1:100 DSHB 4F3), α-PH3 (1:2000 Cell Signaling Technology 9701), α-dpERK (1:250 Sigma Aldrich M-8159), α-Ct (1:50 DSHB 2B10), α-Shotgun (1:50 DSHB DCAD2), α-Arm (1:100 DSHB N2 7A1), α-Wg (1:50 DSHB 4D4), α-Vg (1:200 Gift from Kirsten Guss), α-Twi (1:2000 gift from S. Roth), Phalloidin-iFlor 647 and Phalloidin-iFlor 555 (1:2000 Abcam ab176756 and ab176759, respectively). Alexa Fluor-conjugated secondary antibodies (1:1000, Thermo Fisher Scientific) were used for immunofluorescence detection (see Supplementary Table 3 for details).

L3 larvae were dissected in cold PBS and wing discs together with Tr2 trachea were fixed in 4% formaldehyde. After extensive washing, the samples were permeablized with TritonX-100 and then blocked in 10% donkey serum, and incubated with primary antibodies that had been diluted in blocking buffer. The following primary antibodies were used: α-dpERK (Sigma); α-laminin (from J. Fessler; Fessler et al., 1987 (link)); α-pMad (from E. Laufer and P. ten Dijke) (Persson et al., 1998 (link)); α-Senseless (from H. Bellen), α-Discs large and α-Dally-like (Developmental Studies Hybridoma Bank); α-cleaved Caspase-3 (Asp¹⁷⁵) and α-phosphohistone H3 (Ser¹⁰) (Cell Signaling Technology, Danvers, MA). Secondary antibodies were conjugated to Alexa Fluor 405, 488, 555, or 647. Samples were mounted in Vectashield.