Chicken anti gfp

The following antibodies were used for immunoblot analysis: rabbit-anti-IKK2 (CST-2678, Cell Signaling, Danvers, MA, USA), rabbit-anti-p-p65 (CST-3033, Cell Signaling, Danvers, MA, USA), rabbit-anti-p65 (sc-372, Dallas, TX, USA), chicken-anti-GFP (ab13970, Cambridge, UK), rabbit-anti-tubulin (ab6046, Cambridge, UK), rabbit-anti-PLP1 (CST-28702, Cell Signaling, Danvers, MA, USA), rabbit-anti-MOG (CST-96457, Cell Signaling, Danvers, MA, USA), rabbit-anti-MBP (CST-2396, Cell Signaling, Danvers, MA, USA), rabbit-anti-GAPDH (CST-3686, Cell Signaling, Danvers, MA, USA), rabbit-anti-p-eIF2α (CST-3597, Cell Signaling, Danvers, MA, USA), rabbit-anti-eIF2α (CST-5324, Cell Signaling, Danvers, MA, USA), rabbit-anti-ERK2 (sc-1647, Dallas, TX, USA).
For immunofluorescence, the following primary antibodies were used: mouse-anti-CC1 (OP80, Merck, Darmstadt, Germany), mouse-anti-GSTπ (BD610719, BD, Franklin Lakes, NJ, USA), chicken-anti-GFP (GFP-1020, AvesLab, Davis, CA, USA), mouse-anti-GFAP (sc-33673, Dallas, TX, USA), rabbit-anti-NG2 (AB5320, Merck, Darmstadt, Germany), rabbit-anti-MBP (Biolegend 836,504, San Diego, CA, USA), mouse-anti-Neurofilament-H (SMI32P) (Biolegend 801,701, San Diego, CA, USA), rabbit-anti-ß3-tubulin (Biolegend 802,001, San Diego, CA, USA), rabbit- anti RFP (abcam ab124754, Cambridge, UK).

Larvae were incubated with 10 mM ANL for 24 hr. After incubation, the media was replaced with fresh E3. Larvae were sacrificed on ice-cold water (30 min). Heads were dissected using a scalpel and immediately snap frozen in tubes (1.5 ml, Eppendorf) that were pre-cooled using dry ice and stored at −80C until lysis. Tissue was homogenized and lysed using a pestle in lysis buffer (1% in Triton X100, 0.4% (w/v) SDS in PBS pH 7.8, 1:1000 EDTA free protease inhibitors (Calbiochem) and benzonase (Sigma, 1:1,000), and denatured at 75°C, 13,000 rpm for 10 min. Lysates were then cleared by centrifugation. BONCAT was performed as previously described (Dieterich et al., 2007 (link)). In brief, 60 μg proteins were dissolved in 120 μL PBS pH 7.8 supplemented with 0.01% SDS, 0.1% Triton, 300 μM Triazol (Sigma, 678937), 50 μM biotin-alkyne tag (Thermo, B10185) and 83 μg/mL CuBr (prepared by dilution of fresh 10 mg/mL solution in DMSO) at 4°C overnight in the dark. Biotinylated proteins were then separated by gel electrophoresis and immunoblotted with 1:1000 chicken anti-GFP (Aves), 1:1000 rabbit anti-biotin (Cell signaling) and Donkey anti-chicken IR680, goat anti-rabbit IR800 (IB, 1:10,000, Licor) antibodies.