Hrp conjugated secondary antibody

Fly heads were collected and mashed in a sample lysis buffer. The samples with SDS sample buffer were boiled and run on a 10% SDS-PAGE gel and transferred to nitrocellulose membranes. Primary antibodies for PO₄-CREBB (1:1000), pan-CREBB (1:5000) (Fropf et al., 2013 (link)), CREBA (1:5000) (Andrew et al., 1997 (link)) (Developmental Studies Hybridoma Bank), HA-tag (1:2000) (BioLegend), His-tag (1:2000) (Clontech) and actin (1:10,000) (Abcam) were used and detected by HRP conjugated secondary antibody (1:10,000) (Jackson ImmunoResearch). For detecting the ATG2-CREBB protein (Fropf et al., 2013 (link)), 50 adult brains were dissected and collected in 20 μL of the sample lysis buffer and placed at −80°C for overnight. After vortexing for one minute, samples were mixed with SDS sample buffer and boiled before loading into an SDS-PAGE gel and transferred to nitrocellulose. Primary antibodies for ATG2-CREBB (1:1,000) (Fropf et al., 2013 (link)) was used and detected by HRP conjugated secondary antibody (1:10,000) (Jackson ImmunoResearch). An ECL reagent kit (ThermoFisher) was used for Western blot detection.

For immunoblotting, cell lysates were separated on 8–15% SDS-PAGE and transferred onto PVDF membranes (Merck Millipore, Burlington, MA, USA). The PVDF membranes were blocked with 5% BSA and then incubated with primary antibodies. After washing with PBST, the membranes were incubated with HRP-conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA, USA). The specific signal was detected using ECL reagents (Merck Millipore) and a UVP imaging system (Upland, CA, USA). For immunohistochemical staining, tumor tissues were fixed in 10% buffered formalin and paraffin-embedded sections were prepared. The sections were de-paraffinized in xylene, then dehydrated in ethanol and finally rehydrated in distilled water. After antigen retrieval, the sections were blocked with 5% BSA and then incubated with primary antibodies overnight. Then, HRP-conjugated secondary antibodies (Jackson Immunoresearch) and AEC reagent (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were used to detect specific signals. The primary antibodies used for immunoblotting were Oct4 (Cell Signaling Technology, Beverly MA, USA), Stat1 (Santa Cruz), phospho-Stat1 (Tyr701) (Cell Signaling Technology), Mcl-1 (Santa Cruz Biotechnology, Inc.), cleaved caspase-3 (Cell Signaling Technology) and β-actin (Sigma-Aldrich, St. Louis, MO, USA).

Protein samples were separated on 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride membranes (PVDF) (Millipore, Billerica, MA) for protein bands detection. The membranes were blocked in 5% nonfat milk. Primary antibodies including anti-GAPDH (1:2000, Abmart, Shanghai, CHN), anti-BCL2 (1:500, Abcam, Cambridge, UK), anti-Caspase 9 (1:1000, Cell Signaling Techonolgy, MA, USA), anti-cleaved-Caspase 9 (1:500, Cell Signaling Techonolgy, MA, USA), anti-Caspase 3 (1:1000, Abcam, Cambridge, UK), and anti-cleaved-Caspase 3 (1:1000, Abcam, Cambridge, UK) were used for incubation overnight at 4 °C. The membranes were incubated with HRP-conjugated secondary antibody (1:3000, Jackson Immuno Research, PA, USA) at room temperature for 1 h and the protein bands were detected by Pierce Fast Western Blot Kit (Thermo Fisher Scientific, MA, USA).