Journal: eLife
Article Title: CLUH controls astrin-1 expression to couple mitochondrial metabolism to cell cycle progression
doi: 10.7554/eLife.74552
Figure Lengend Snippet: ( A, B ) Western blots of reciprocal co-IPs of endogenous CLUH, astrin, and kinastrin in HeLa cells. Two different antibodies have been used to pull down CLUH (1, 2). Asterisk marks IgG light chain. ( C ) Scheme of human SPAG5 cDNA with indicated UTRs and ORFs. Closeup shows positions of the uATG and ATG1–6 with surrounding Kozak sequences. ( D, E ) Western blots of HeLa cells overexpressing FLAG-tagged astrin constructs. Pan-actin was used as loading control. ( F ) Western blots of reciprocal co-IPs of endogenous CLUH and overexpressed FLAG-tagged astrin full length (ATG1-SPAG5) or a N-terminal deleted variant (Δ151-SPAG5) in WT and CLUH KO HeLa cells. Pan-actin was used as loading control for input samples. Asterisks indicate additional astrin bands appearing upon overexpression of the N-terminal deleted variant. ( G ) Confocal immunofluorescence pictures of HeLa cells overexpressing FLAG-tagged astrin-1 and astrin-2 (SPAG5), astrin-1 (ATG3 + 4 + 5 GGG -SPAG5) or astrin-2 (ATG1 GGG -SPAG5) alone stained for FLAG (green) and CLUH (red). DAPI was used to stain nuclei (blue). Small boxes in left corners show a ×4 magnified area of boxed regions. Scale bar, 10 µm. ( H ) Polysome profiling of HeLa cells chemically crosslinked with dithiobis succinimidyl propionate (DSP). At the top, absorbance profile at 254 nm of the fractions is shown with indicated peaks of 40S and 60S ribosomal subunits, 80S monosome and polysomes; at the bottom the corresponding western blots of the fractions are shown. RPL7 was used as a marker for ribosomes. Figure 1—source data 1. Uncropped blots for . Figure 1—source data 2. Uncropped blots for . Figure 1—source data 3. Unedited blots for .
Article Snippet: The following primary antibodies were used for western blotting: rabbit polyclonal anti-CLUH antibodies [detecting human CLUH; #NB100-93305 (1), #NB100-93306 (2)], rabbit polyclonal anti-RPS6 (#NB100-1595), rabbit polyclonal anti-RPL7 (#NB100-2269) antibodies from Novus Biologicals; rabbit polyclonal anti-astrin (#14726-1-AP) antibody from ProteinTech; rabbit polyclonal anti-FLAG (#F7425) and mouse monoclonal anti-FLAG (#F3165) antibodies from Sigma-Aldrich; mouse monoclonal pan-actin (#MAB1501) and anti-GAPDH (#MAB374) antibodies from EMD Millipore; rabbit polyclonal anti-CLUH antibody (detecting murine CLUH; #ARP70642_P050) from Aviva; rabbit polyclonal anti-kinastrin (#ab122769) and rabbit polyclonal pH3-Ser10 (#ab5176) antibodies from Abcam; mouse monoclonal anti-SDHA (#459200), anti-NDUFA9 (#459100), and anti-UQCRC1 (#459140) from Molecular probes; rabbit polyclonal pRB1-Ser807/811 (#9308), rabbit monoclonal pCDK1-Tyr15 (#4539), mouse monoclonal anti-cyclin D3 (#2936), and rabbit polyclonal pRPS6-Ser235/236 (#2211) antibodies from Cell Signaling and mouse monoclonal anti-CDK1 (#sc-54) antibody from Santa Cruz Biotechnologies.
Techniques: Western Blot, Construct, Control, Variant Assay, Over Expression, Immunofluorescence, Staining, Marker
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