Mouse anti dmyc

Mobility shift of phosphorylated dMyc protein was detected by phosphate affinity SDS-PAGE using acrylamide-pendant phos-tag (Phos-tag AAL-107). Briefly, 50–100 μM phos-tag acrylamide and 100–200 μM MnCl₂ were added to normal 6% polyacrylamide gel. After electrophoresis, the gel was washed with transfer buffer containing 1 mM EDTA for 10 min with gentle agitation, and then with transfer buffer without EDTA for 10 min. Proteins were transferred to Immobilon-FL transfer membranes (Millipore). For western blotting, pupae were homogenized in SDS sample buffer with a pellet pestle (Kimble-Kontes), and the proteins were fractionated using SDS-PAGE. The proteins were transferred to Immobilon-FL transfer membranes in Tris-glycine buffer. The blots were probed with mouse anti-HA 1:500 (Cell Signaling), mouse anti-dMyc (1:50), and rabbit anti-α tubulin 1:15000 (Sigma), and subsequently with IRDye 800-labeled anti-mouse IgG and IRDye 680-labeled anti-rabbit IgG (Licor). Signals were detected using an Odyssey infrared imaging system.

The following flies were used in this study: brm^T362 is from J Treisman; erm¹, erm², UAS-Erm^CTHA, UAS-Brm (AK Dingwall), 9D11-Gal4 (erm-Gal4; GM Rubin). brm², bap55^LL05905, Erm RNAi (#26778; BDSC) are from Bloomington Drosophila stock center. VDRC RNAi lines used: Brm (GD37720 and 37721GD), Bap60 (KK103634), Snr1 (KK108599, GD12645, and BDRC#32372), Bap55 (GD24704), Moira (GD6969), Bap180 (KK108618), dMi-2 (KK107204), nurf301 (GD46645), Acf1 (GD33446) and ISWI (GD24505). The type II neuroblast driver: w; UAS-Dicer 2, wor-Gal4, ase-Gal80/CyO; UAS-mCD8-GFP/TM3, Ser (Neumuller et al., 2011 (link)).
The primary antibodies used were: guinea-pig anti-Dpn (1:1000, J Skeath), anti-Insc (1:1000); rabbit anti-aPKCζ C20 (1:100; Santa Cruz Biotechnologies, Dallas, TX); guinea-pig anti-Numb (1:1000; J Skeath); mouse anti-Mira (1:50; F Matsuzaki); rat anti-CD8 (1:250; Caltag laboratories, United Kingdom); rabbit anti-GFP (1:500; Molecular Probes, Eugene, OR); rabbit anti-Asense (1:1000; YN Jan); rabbit anti-PntP1 (1:100; J Skeath); rabbit anti-Brm (1:100; L Zhang); rat anti-phospho-Histone H3 (1:1000; Cell Signaling, Danvers, MA); rabbit anti-phospho-Histone H3 (1:200; Sigma, St Louis, MO); mouse anti-dMyc (1:5; B Edgar). Antibodies for western blotting used were: mouse anti-Myc (1:2000; Abcam, United Kingdom) and mouse anti-Flag (1:1000; Sigma).