Western Blot:Article Title: Ribosome profiling reveals a functional role for autophagy in mRNA translational control.
Article Snippet: HEK293Ts were purchased from ATCC (CRL-3216). .. Antibodies used for immunoblotting are anti-BRCA2 rabbit pAb from Bioss USA bs1210R (1:300); anti-p62 guinea pig pAb from Progen GP-62-C; anti-LC3 mouse pAb, Fung et al.64, now commercially available from EMD Millipore ABC232; anti-phospho-S6 S240/244 rabbit mAb Cell Signaling #2215; anti-S6 rabbit mAb Cell Signaling #2217; anti-α-tubulin rabbit mAb Cell Signaling #2125BC; anti-phospho-4EBP1 S65 rabbit pAb Cell Signaling #9451; anti-4EBP1 rabbit pAb Cell Signaling #9452; anti-GAPDH mouse mAb Millipore MAB374; anti-phospho-eIF2α S51 rabbit pAb Cell Signaling #9721; anti-eIF2α S51 rabbit pAb Cell Signaling #9722; anti-eIF4G1 rabbit mAb Cell Signaling #2469; anti-eIF4G2 rabbit pAb Cell Signaling #2182; anti-eIF4E rabbit pAb Cell Signaling #9742; anti-eIF4E2 rabbit pAb Pierce #PA5-11798; anti-Atg12 (mouse specific) rabbit pAb Cell Signaling #2011BC; anti-Atg5 rabbit pAb Novus Biologicals NB110-53818; anti-Atg7 rabbit pAb Cell Signaling #2631; anti-Atf4 rabbit mAb Cell Signaling #11815; anti-Cdc25a rabbit pAb Cell Signaling #3652; anti-Cpt2 Abcam ab71435; anti-Pfkfb3 rabbit pAb ABclonal Biotech A6945; anti-eEF2 rabbit pAb Cell Signaling #2332; anti-Mcl-1 rabbit pAb Rockland 800-401-394S; anti-Irf7 rabbit mAb Abcam ab109255; anti-γH2AX S139 mouse mAb Upstate #05-636; anti-cleaved Caspase 3 (Asp175) Rabbit pAb Cell Signaling #9661; anti-eIF4A1 rabbit pAb Cell Signaling #2490; anti-MSI1 rabbit pAb EMD Millipore AB5977 (1:500); anti-GFP mouse mAb Neuromab N86/8; anti-puromycin mouse mAb Kerafast EQ0001. ..
Article Title: Ribosome profiling reveals a functional role for autophagy in mRNA translational control
Article Snippet: Membranes were blocked for 1 h in 5% milk or 5% BSA in PBS with 0.1% Tween 20, incubated in primary antibody (1:1000 unless otherwise specified) overnight at 4 °C, washed, incubated for 1 h at RT with HRP-conjugated goat secondary antibodies (1:5000; Jackson ImmunoResearch Laboratories; streptavidin HRP conjugate Thermo Scientific Pierce 21130), washed, and visualized via enhanced chemiluminescence (Thermo Fisher Scientific). .. Antibodies used for immunoblotting are anti-BRCA2 rabbit pAb from Bioss USA bs1210R (1:300); anti-p62 guinea pig pAb from Progen GP-62-C; anti-LC3 mouse pAb, Fung et al. , now commercially available from EMD Millipore ABC232; anti-phospho-S6 S240/244 rabbit mAb Cell Signaling #2215; anti-S6 rabbit mAb Cell Signaling #2217; anti-α-tubulin rabbit mAb Cell Signaling #2125BC; anti-phospho-4EBP1 S65 rabbit pAb Cell Signaling #9451; anti-4EBP1 rabbit pAb Cell Signaling #9452; anti-GAPDH mouse mAb Millipore MAB374; anti-phospho-eIF2α S51 rabbit pAb Cell Signaling #9721; anti-eIF2α S51 rabbit pAb Cell Signaling #9722; anti-eIF4G1 rabbit mAb Cell Signaling #2469; anti-eIF4G2 rabbit pAb Cell Signaling #2182; anti-eIF4E rabbit pAb Cell Signaling #9742; anti-eIF4E2 rabbit pAb Pierce #PA5-11798; anti-Atg12 (mouse specific) rabbit pAb Cell Signaling #2011BC; anti-Atg5 rabbit pAb Novus Biologicals NB110-53818; anti-Atg7 rabbit pAb Cell Signaling #2631; anti-Atf4 rabbit mAb Cell Signaling #11815; anti-Cdc25a rabbit pAb Cell Signaling #3652; anti-Cpt2 Abcam ab71435; anti-Pfkfb3 rabbit pAb ABclonal Biotech A6945; anti-eEF2 rabbit pAb Cell Signaling #2332; anti-Mcl-1 rabbit pAb Rockland 800-401-394S; anti-Irf7 rabbit mAb Abcam ab109255; anti-γH2AX S139 mouse mAb Upstate #05-636; anti-cleaved Caspase 3 (Asp175) Rabbit pAb Cell Signaling #9661; anti-eIF4A1 rabbit pAb Cell Signaling #2490; anti-MSI1 rabbit pAb EMD Millipore AB5977 (1:500); anti-GFP mouse mAb Neuromab N86/8; anti-puromycin mouse mAb Kerafast EQ0001. .. In total, 200,000 cells were grown in 6-well plates and incubated for 1.5 h DMEM lacking methionine, DMEM lacking methionine and glucose, DMEM lacking methionine and glutamine (UCSF cell culture facility) or HBSS (Gibco), upon which 30 μCi of exogenous 35-S L-Methionine (Perkin Elmer NEG009A001MC) was added for the last 30 min.
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