Mca78g

Oocytes were fixed for 30–60 min at 37 °C in 100 mM HEPES (pH 7; titrated with KOH), 50 mM EGTA (pH 7; titrated with KOH), 2% formaldehyde (methanol-free) and 0.2% Triton X-100, based on previously published methods51 (link). Fixed oocytes were incubated in PBS with 0.1% Triton X-100 overnight at 4 °C. Antibody incubations were performed in PBS, 3% BSA and 0.1% Triton X-100. Primary antibodies used were mouse anti-pericentrin (30, BD Biosciences 611815; 1:750), mouse anti-γ-tubulin (GTU88, Sigma T6557; 1:3,000), rat anti-tyrosinated-α-tubulin (YOL1/34, AbD Serotec MCA78G; 1:3,000) and mouse anti-PLK1 (35–206, Abcam Ab17056; 1:1,000). Secondary antibodies used were Alexa-Fluor-488-labelled anti-mouse (Molecular Probes A11029; 1:400) and Alexa-Fluor-647-labelled anti-rat (Molecular Probes A21247; 1:400). DNA was stained with 5 mg ml⁻¹ Hoechst 33342 (Molecular Probes H3570).

Mouse anti-Lamin C (DSHB, no. LC28.26-c) was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa. Rat anti-α-Tubulin (1:200 dilution; Bio-Rad MCA78G), chicken anti-GFP (1:200 dilution; Abcam #13970), rabbit anti-BAF [1:300 dilution; provided by Paul Fisher, Stony Brook, NY, and Ryszard Rzepecki, University of Wroclaw, Poland (Furukawa et al., 2003 (link))] and rat anti-E2F1 [1:200 dilution; provided by Stefan Thor, University of Queensland, Australia (Baumgardt et al., 2014 (link)) and Jonathan Benito-Sipos, University of Madrid] were used for immunofluorescence. Rabbit anti-Otefin (1:300 dilution) was provided by Y. Gruenbaum (Hebrew University, Jerusalem, Israel). Secondary antibodies used were Alexa Fluor 488, 555 and 647 conjugated secondary antibodies against rat, chick, rabbit and mouse (1:300 dilution), purchased from The Jackson Laboratory and ThermoFisher Scientific.
For labeling of chromatin, we used DAPI (1 µg/ml; Sigma-Aldrich). For F-actin labeling, we used TRITC-phalloidin (Sigma-Aldrich P1951). Labeling was performed by exposing the larvae from experimental and control groups to a similar antibody mix in the same tube.

The following antibodies used in this study were purchased from Cell Signaling: acetyl-α-tubulin (1:1000; #5335), TAB1 (1:1000; #3226), pERK (1:2000; #9101), total ERK (1:2000; #4695), pAKT-S473 (1:1000; #4060), pP38 (1:2000; #4511), pJNK (1:2000; #9255), cMYC (1:2000; #13987), pGSK3β-Ser9 (1:2000; #5558), total GSK3β (1:2000; #12456), pTAK1 (1:1000; #9339), total TAK1 (1:1000; #4505), total AKT (1:1000; #4691), pPRAS40 (1:2000; #2997), total PRAS40 (1:2000; #2610), pSmad2 (1:1000; #8828), and Myc-Tag (1:4000; #2276). Total tubulin YOL1/34 (1:2000; MCA78G) (Abcam), αHA (1:3000; #11867431001), α-Flag (1:4000; F1804) (Sigma), αTAT1 phosphoserine 237 (1:250) (Thermo Scientific), and αTAT1 (1:500; sc-167354) (Santa Cruz) were also used.