Polyclonal anti flag

293T whole cell extracts and mouse liver lysates were prepared using Lysis buffer containing 1%TX-100 as previously described (Lamia et al., 2004 (link)). MEF cell extracts were prepared from RIPA buffer containing 1% TX-100, 147 mM NaCl, 12 mM sodium deoxycolate, 0.1% SDS, 50 mM Tris pH 8.0, 10 mM EDTA, 50 μM PMSF, phosphatase inhibitors (cat #P5266 and cat #P0044; Sigma, St. Louis, MO) and protease inhibitor (cat #11697498001; Roche, Switzerland). For ubiquitination experiments, iodoacetamide was added to the buffer to a final concentration of 5 mM (Fisher AC122270050).
Antibodies were anti-Flag M2 agarose beads, anti-Flag polyclonal, anti-v5 polyclonal, anti-Lamin A, anti-aTubulin, and anti-βactin from Sigma, anti-Hausp and anti-V5 from Bethyl Labs (Montgomery, TX, cat #A300-033A and cat #A190-120A), anti-53BP1 from Novus Biologicals (Littleton, CO, NB100-304), Cry1-CT and Cry2-CT as described (Lamia et al., 2011 (link)), anti-p21 from Santa Cruz Biotechnologies (Dallas, TX, cat #sc-6246), anti-p53 as previously described (Pasini et al., 2004 (link)), and anti-Ubiquitin, anti-phospho-P53 (S15), and anti-phosphoATM/ATR substrate (phospho-SQ/TQ) from Cell Signaling Technology (Danvers, MA). Anti-Cry1-phosphoS588 antibody was affinity purified from rabbit antisera raised against a phospho-S588 containing peptide.

293 T whole cell extracts were prepared using lysis buffer containing 1%TX-100, 50 mM HEPES (pH 7.4), 138 mM KCl, 4 mM NaCl, 50 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 1 mM EGTA pH 8, 50 μM PMSF, 1 mM β-Glycerophosphate, 1 mM sodium orthovanadate, and protease inhibitors (Roche cat # 11697498001 or Thermo Scientific cat # 88265) as previously described (Lamia et al.^{40 (link)}). ASF cell extracts were prepared from RIPA buffer containing 1% TX-100, 147 mM NaCl, 12 mM sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0, 10 mM EDTA, 50 μM PMSF, 1 mM β-Glycerophosphate, 1 mM sodium orthovanadate, 1 mM sodium fluoride, and protease inhibitors (Thermo Scientific cat # 88265). Antibodies used for immunoprecipitation were anti-FLAG M2 agarose beads (Sigma cat # A2220) and monoclonal anti-HA agarose beads (Sigma cat # A2095). Antibodies for Western blot were anti-FLAG polyclonal (Sigma cat # F7425), anti-HA polyclonal (Sigma cat # H6908), anti-MYC polyclonal (Sigma cat # C3956), anti-SKP1 (BD Biosciences cat # BDB610530), anti-CUL1 (Life Technologies cat # 71–8700), anti-αTubulin (Sigma cat # T5168), anti-βactin (Sigma cat # A1978), anti-AFG3L2[N1N2] (GeneTex cat # GTX102036), and CRY1-CT and CRY2-CT as described (Lamia et al., 2011).

Cell lysates were separated by SDS/PAGE, and proteins were transferred on to nitrocellulose membranes. The membranes were blocked with TBST (20 mM Tris [pH 7.5], 150 mM NaCl, and 0.1% Tween 20) containing 5% skim milk for 1 h. The blot was then incubated with primary antibody at 4°C overnight and washed thrice with TBST. This was followed by incubating with secondary antibody for 1 h, washed again with TBST, and finally developed through enhanced chemiluminescence. The primary antibodies used in the study included mAb 7H6 (as described above), anti-GAPDH polyclonal (Santa Cruz Biotechnology), and anti-FLAG polyclonal (Sigma) antibodies. Secondary antibodies used were HRP-conjugated goat anti-mouse and goat anti-rabbit IgG (Bio-Rad).

pCI/Dyrk1A and its deletion mutants were constructed and confirmed by Sanger sequencing. pCI/Dyrk1As were tagged with FLAG at the C‐terminus. pCEP4/ASF‐HA, pCEP4/SC35‐HA, and pCEP4/SRp55‐HA were kind gifts from Dr. Tarn of the Institute of Biomedical Sciences, Academia Sinica, Taiwan. The Bcl‐x mini‐gene, comprising Bcl‐x exons 2 and 3 and intron 2 was a gift from Dr. Jianhua Zhou of the University of Massachusetts Medical School. pCEP4/ASF_S3A‐HA, ASF was mutated at Ser‐227, Ser‐234, and Ser‐238 to three Ala, as described previously.
²⁵ (link) Small inference RNAs (siRNAs) of Dyrk1A and ASF, and Monoclonal anti‐Bcl‐xL were obtained from Santa Cruz Biotechnology. Monoclonal antibody 8D9 was raised against a histidine‐tagged protein containing the first 160 residues of rat Dyrk1A.
³¹ (link) The monoclonal anti‐HA, polyclonal anti‐FLAG, and monoclonal anti‐Dyrk1A (N‐terminal) were purchased from Sigma. Polyclonal Bcl‐xS, and monoclonal anti‐NeuN were purchased from Thermo Fisher Scientific. Horseradish peroxidase (HRP) conjugated secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Enhanced chemiluminescence ECL kit was bought from Thermo Fisher Scientific.

The following antibodies were obtained commercially: anti-γ-tubulin, polyclonal anti-FLAG, anti-Cyclin A, monoclonal anti-FLAG M2, anti-α-tubulin and anti-acetylated-α-tubulin (all from Sigma, St. Louis, MO), anti-Cyclin E, anti-CDK2 phospho-Thr160, anti-Akt and anti-Akt phospho-Thr308 (Cell Signaling, Beverly, MA), anti-centrin 20H5 (Millipore, Billerica, MA), polyclonal anti-β-catenin and anti-GSK3β (Abcam, Cambridge, UK), anti-Ku70 (Genetex, Trvine, CA), anti-DNA-PKcs, and anti-DNA-PKcs phospho-Thr 2609 (Santa Cruz Biotech, Santa Cruz, CA). The immune sera against SF-1 have been described previously [19 (link)].

To obtain whole cell extract, NSPCs were harvested in RIPA buffer containing 320 mM sucrose, 50 mM TRIS pH 7.5, 1% Triton, 10% glycerol and 1% of inhibitor of proteases cocktail (Sigma-Aldrich), incubated on ice for 30 min and centrifuged for 12 min at 13,000× g. Protein amount was quantified by Bradford assay (BioRad, Hercules, CA, USA) and 10 or 20 µg of protein was subjected to SDS-polyacrylamide gel electrophoresis and transferred overnight onto polyvinylidene fluoride membrane (Immobilion-PSQ; Millipore) [82 (link)]. Membranes were blocked in 5% non-fat dry milk and then incubated with the following primary antibodies: monoclonal anti-FUS (1:750; Santa-Cruz), polyclonal anti-FLAG (1:750; Sigma-Aldrich), monoclonal anti-PCNA (1:750; Sigma-Aldrich), polyclonal anti-Cdk2 (1:400, Santa Cruz), polyclonal anti-DCX (1:200, Abcam, Cambridge, UK), and polyclonal anti-PAX6 (1:200; BioLegend). Blots were washed and incubated with the appropriate horseradish peroxidase secondary antibodies (1:10.000, Jackson Immunoresearch). Blots were then washed again, incubated in lumi-light enhanced chemiluminescence substrate (BioRad) and exposed to chemidoc (BioRad). Densitometric analysis on scanned blots was performed using the ImageLab program (BioRad).

The anti-peptide polyclonal antibody raised against the C-terminal region of the human a-SMN (#910) was prepared in rabbits in 2005 by NeoMPS (Strasbourg, France; diluted 1:500 for IF). The specific antibody samples used in this study have been previously described [13 (link)]. Mouse anti-tag (anti-Xpress), revealing transfected proteins only, was purchased from Life Technologies (diluted 1:500 for WB and IF experiments), polyclonal anti-Flag from Sigma-Aldrich (diluted 1:500 for IF); anti-SMN clone 8 from BD Transduction Laboratories (diluted 1:20,000 for WB); mouse anti-actin from Millipore (diluted 1:5,000 for WB); mouse anti-neurofilament 200 (NF-200) from Sigma-Aldrich (diluited 1:500 for IF).