Rabbit anti dperk

Western blot was performed as described previously (Tien et al., 2015 (link)). Briefly, RNAs encoding tagged proteins were injected into 2- to 4-cell stage embryos. Embryonic lysate was obtained at gastrula stages by lysing embryos with cold buffer containing 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1mM EDTA, 10% glycerol and 0.5% Triton X-100. The lysate was either loaded on the SDS–PAGE directly (for the dpErk assay) with the 2× SDS gel-loading buffer (100 mM Tris.Cl, pH6.8, 200mM DTT, 4% SDS, 0.2% bromophenol blue, 20% glycerol) or subjected to the co-IP assay. Western blot was performed using rabbit anti-Flag (1:3000, Sigma), rabbit anti-HA (1:3000, Cell Signaling Technology), rabbit anti-dpErk (1:4000, Cell Signaling Technology), rabbit anti-GFP (1:3000, Cell Signaling Technology), or mouse anti-p-threonine (1:2000, Cell Signaling Technology) antibodies. Quantification was performed using NIH ImageJ software. The Student’s t-test was used to assess the statistical significance in differences between YWHAZ and YWHAZ(S230W) in dose-response experiments shown in Figure 4.

Third instar larvae were dissected in 1xPBS and fixed in 4% paraformaldehyde (PFA) for 20 min and stained. For dpERK staining, third instar larvae were dissected in cold 1xPBS and immediate fixed in 8% PFA for 20 min and followed with 10 min ice-cold ethanol treatment in −20 °C before further step. Samples were incubated with primary antibodies at the following dilutions: chicken anti-GFP (1:1000, Abcam, ab13970), rabbit anti-phospho-Histone H3 (Ser10) (1:200, Cell signaling, #9701), rabbit anti-Dcp1 (1:100, Cell signaling, #9578), mouse anti-WASH (1:5, Developmental Studies Hybridoma Bank(DSHB), P3H3) mouse anti-MMP1 (1:50, DSHB, cocktail 1:1:1 of 5H7B11, 3B8D12), mouse anti-Ptp10D (1:50, DSHB, cocktail 1:1 of 8B22F5 and 45E10), rabbit anti-aPKCζ (C-20) (1:250, Santa Cruz Biotechnology (SCBT), sc-216,), mouse anti-dEGFR (1:100, Sigma, E2906), guinea pig anti-capicua⁴¹ (1:1000, a kind gift from Edgar’s Lab), Alexa Fluor^TM Phalloidin 647 (1:50, Thermo Fisher, A22287), rabbit anti-ß-galactosidase (1:150, Cappel Laboratories, #0855976), rabbit anti-dpERK (1:200, cell signaling, #4370) guinea pig anti-DIAP1^{59 (link)} (1:200, a kind gift from Meier Pascal’s lab).

The intestines from female adults were dissected in cold PBS and fixed immediately in PBS containing 4% (weight/vol) paraformaldehyde. Samples were then washed in PBS with 0.1% Triton X-100 (vol/vol) three times, blocked in PBTB [PBT containing 5% (vol/vol) normal goat serum], and incubated with primary antibodies overnight. The following primary antibodies were used: rabbit anti-Clbn (1:400), mouse anti-Prospero (1:200, Developmental Studies Hybridoma Bank), rabbit anti-Pdm1 (1:400), mouse anti-Armadillo (1:50, Developmental Studies Hybridoma Bank), rabbit anti-β-galactosidase (1:200, Promega), rabbit anti-phospho–histone-H3 (1:200, Millipore), rabbit anti-H2AvD pS137 (1:200, Rockland), rabbit anti-dpERK (1:200, Cell Signaling Technology), Alexa-Fluor-555-conjugated Phalloidin (1:500, Thermo Fisher Scientific). After three washes with PBT, secondary anti-rabbit or anti-mouse fluorescence antibodies, including Alexa 488 and 555 (1:400, Cell Signaling Technology), were used. Samples were mounted and analyzed on a Olympus FV1000 confocal laser-scanning microscope. The images were processed using Adobe Photoshop, Illustrator, and ImageJ.

The following antibodies used were: rabbit anti-aPKC (1:500; Santa Cruz); rat anti-crb (1:500; gift from E.Knust); mouse anti-Dlg (1:500; Hybridoma Bank); rabbit anti-dpERK (1:200; Cell signalling); goat anti-GFP (1:500; Abcam); rabbit anti-RFP (1:300; Life Technologies); Phalloidin TRITC (1:200; Sigma Aldrich); mouse anti-Patched (1:100; Hybridoma Bank); rat anti-Srp (1:500 made in the Casanova lab). The TUNEL assay was performed using the In situ Cell Death Detection Kit (Roche). Cy2, Cy3 and Cy5-conjugated secondary antibodies were from Molecular Probes and were used at 1:200 dilutions, and discs were mounted in Vectashield containing DAPI. Confocal images were acquired with a Leica SP5. Images were analysed with Fiji software [National Institutes of Health (NIH) Bethesda, MD] and assembled into figures using both Fiji and the Adobe Photoshop software.