Mouse α β actin

Protein samples for the cap-affinity assay were separated by 8-15% gradient SDS-PAGE (polyacrylamide gel electrophoresis). Ten or twelve percent SDS-PAGE was used for PARP and eIF4E proteins, respectively. Following protein transfer to PVDF (Amersham Biosciences) the membranes were blocked in 5% non-fat dry milk for 1 hour at room temperature in Tris-buffered saline-Tween (TBST: 0.15 M NaCl; 0.01 M Tris-HCl, pH 7.6; 0.05% Tween 20). The membranes were then incubated for 1 hour at ambient temperature or overnight at 4°C with the chosen primary antibody. The primary antibodies employed were rabbit α-eIF4E (catalog number 9742), rabbit α-PARP (catalog number 9542), α-SV40 large T antigen (catalog number 15729) all from Cell Signaling Technology (Danvers, MA) at a 1:1000 dilution, mouse α-β-actin (Sigma-Aldrich, catalog number A1978) at a 1:10,000 dilution and rabbit α-eIF4GI (kindly provided by Nahum Sonenberg, McGill University Montreal, QC, Canada) at a 1:2500 dilution. Preceding and following incubation with the appropriate horseradish peroxidase labeled secondary antibodies, the blots were washed three times for 5 minutes in TBST and detection was then performed utilizing ECL Plus Western Blotting System (Amersham Biosciences) to visualize the bands of interest.

HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal calf serum (FCS, Bodinco DV), 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine. BHK-21 cells were cultured in Glasgow’s minimal essential medium (GMEM, Gibco) supplemented with 8% (v/v) FCS, 10% (v/v) tryptose phosphate broth (TPB), 10 mM HEPES pH 7.4, 100 U/ml penicillin, and 100 mg/ml streptomycin. MARC-145 were cultured in DMEM supplemented with 8% (v/v) FCS, 100 U/ml penicillin, and 100 mg/ml streptomycin. DMEM and cell culture supplements were obtained from Lonza.
Primary antibodies that were used were mouse-α-β-actin (A5316, Sigma-Aldrich), rabbit-α-GFP [9 (link)], mouse-α-V5 (37–7500, Invitrogen), mouse-α-myc (9E10, Roche), mouse-α-HA (ab18181, Abcam), and mouse-α-FLAG (F1804, Sigma-Aldrich). As secondary antibodies, goat-α-mouse-HRP (P0447, Dako), swine-α-rabbit-HRP (P0217, Dako), goat-α-mouse-AlexaFluor488 (A11001, Life Technologies), and donkey-α-rabbit-Cy3 (711-165-152, Jackson). The antibody α-ORF7 (P3-05-A27, MSD Animal Health) was obtained using mouse antiserum. The antibody α-nsp2/3 LV was obtained using rabbit antiserum [13 (link)].

Rabbit α-WRAP53-C2 (cat. no. PA-2020-100, Innovagen AB, Lund, Sweden, used for western blotting), mouse monoclonal α-WDR79 (clone 1F12, cat. no. H00055135-M04, Abnova (VWR International, Stockholm, Sweden), used for immunofluorescence), rabbit α-WRAP53 (cat. no. HPA023026, Atlas Antibodies, used for immunohistochemistry), mouse α-γH2AX (cat. no. 05-636, Millipore, Solna, Sweden), rabbit α-γH2AX (cat. no. 2577, Cell Signaling, Bionordika, Stockholm, Sweden), rabbit α-MDC1 (cat. no. ab11169, Abcam, Cambridge, UK), rabbit α-RNF168 (cat. no. ABE367, Millipore), mouse α-conjugated ubiquitin (FK2; cat. no. ST1200, Calbiochem, Millipore), rabbit α-53BP1 (cat. no. NB100-904, Novus Biologicals, Bio-Techne, Abingdon, UK), mouse α-BRCA1 (cat. no. sc-6954, Santa Cruz Biotechnology, Heidelberg, Germany), rabbit α-RAD51 (cat. no. sc-8349, Santa Cruz Biotechnology), mouse α-β-actin (Sigma-Aldrich, Stockholm, Sweden), mouse HSP90 α/β (cat. no. sc-13119, Santa Cruz Biotechnology) and rabbit lamin A/C (cat. no. sc-20681, Santa Cruz Biotechnology) were all used in this study.

Primary antibodies used were as follows: rabbit α-TXNDC11 (Abcam, ab188329; 1:5,000), rabbit α-FLAG (Sigma-Aldrich, F7425; 1:10,000), mouse M1 α-FLAG (Sigma-Aldrich, F3040; 1:10,000), rabbit α-EDEM2 (Sigma-Aldrich, E1728; 1:5,000), rabbit α-EDEM3 (Sigma-Aldrich, E0409; 1:5,000), rabbit α-PDI (Cell Signaling, #2446; 1:5,000), rabbit α-GANAB (GeneTex, GTX102237; 1:5,000), rabbit α-SEL1L (Santa Cruz Biotechnology, sc-48080; 1:2,000), mouse α-calnexin (AF8, a kind gift from M. Brenner, Harvard Medical School; 1:10,000) and mouse α-β-actin (Sigma-Aldrich, A5316; 1:10,000).

Cortex and heart tissues were homogenized, protein extracted, and immunoblotting performed following standard protocols. Total protein was quantified using BCA Protein Assay Kit (Thermo Scientific), as per manufacturer's instructions, and 50 μg total protein was loaded per sample. The following primary antibodies were employed: rabbit α−NF-κB P65 (1:1,000, Cell Signaling Ab#8242) and rabbit α−Phospho-NF-κB P65 Ser536 (1:1,000; Cell Signaling Ab#3033); mouse α-β-actin (1:2,000; Sigma A5441). Appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies were from Pierce, and signal was detected with SuperSignal West Pico Chemiluminescent Substrate (Pierce), and a FluorChem M imager (ProteinSimple). Even loading was confirmed by β-actin, and densitometry was performed on total p65 and phospho-p65 bands using ImageJ (http://rsb.info.nih.gov/ij/index.html) from three pairs of wild-type and three Mecp2-null male littermates. Images have been cropped for presentation. Full-size images are presented in Supplementary Fig. 5. Data were assessed by unpaired two-sided t-test. Error bars indicate s.e.m.

Protein samples were separated by 10% SDS-PAGE (polyacrylamide gel electrophoresis) or by 8-15% gradient gels. After protein transfer to PVDF (polyvinylidene difluoride), the membranes were blocked in 5% non-fat dry milk for 1 hour at room temperature in Tris-buffered saline-Tween (TBST: 0.15 M NaCl; 0.01 M Tris-HCl, pH 7.6; 0.05% Tween 20). Membranes were then incubated for 1 hour at ambient temperature or overnight at 4o C with the chosen primary antibody. The primary antibodies employed were rabbit α-HSP70 antibody (Cell Signaling), rabbit α-PARP antibody (Cell Signaling), each at a 1:1000 dilution, and mouse α-β-actin (Sigma) at 1:10,000 dilution. Before and after incubation with the appropriate horseradish peroxidase labeled secondary antibodies, the blots were washed three times for 5 minutes in TBST. Detection was performed utilizing ECL Plus Western Blotting System (Amersham Biosciences) to visualize the bands of interest. Protein band density was measured by using ImageJ, a public domain java image processing program.