representative western blot  (Wolters Kluwer Health)

Journal: EMBO Reports

Article Title: ChemRAP uncovers specific mRNA translation regulation via RNA 5′ phospho-methylation

doi: 10.1038/s44319-024-00059-z

Figure Lengend Snippet: ( A ) HeLa-S3-FlpIn Control and BCDIN3Df (BCDIN3D-FLAG) lysates were treated with mock or 30 µg RNase A prior to FLAG co-immunoprecipitation and elution with a FLAG peptide. Inputs and FLAG eluates were analyzed by western blots with the indicated antibodies. Equal co-immuno-precipitation of BCDIN3D was verified by Coomassie staining. ( B ) Direct comparison of BCDIN3D binding to GST-EPRS and GST-MARS (See complete analysis in Appendix Fig. ). ( C ) FLAG eluates from HeLa-S3-FlpIn ± EPRSf (EPRS-FLAG) or HeLa-S3-FlpIn-BCDIN3D-KO ± EPRSf were analyzed by LC-MS/MS. Plotted is the mean from n = 2 independent biological replicates of the Percentage of Total Spectra (PTS) for each protein normalized to EPRS PTS and HeLa-S3-FlpIn-EPRSf. ( D ) Quantitative LI-COR western blot analysis with antibodies against EPRS (red) and MARS (green) of input and anti-GFP or anti-EPRS immune-precipitates of HeLa-S3-FlpIn Control and BCDIN3D-KO cells. Below the western blot is shown the ratio of MARS/EPRS normalized to control. ( E ) Enrichment of GAIT subunits (EPRS, GAPDH, NSAP1 and RPL13A) in HeLa-S3-FlpIn and HeLa-S3-FlpIn-BCDIN3D-KO: -(Control) and -EPRSf FLAG eluates. Plotted is the mean from n = 2 independent biological repeats of the PTS for each protein. ( F ) GST pulldown with GST-MARS assessing binding of untagged recombinant EPRS in the absence or presence of RNA-5′-P or 5′-Pme. ( G ) Northern blot analysis of MARSf and EPRSf interacting RNAs. The bottom panel shows the SYBR-Gold-stained gel used for the northern blots on the top. .

Article Snippet: Figure 6 BCDIN3D regulates LRPPRC translation. ( A ) Representative quantitative LI-COR western blots with antibodies against LRPPRC (green) and β-Tubulin (red) of whole-cell extracts collected after 48 h of reverse transfection of siNC and siLRPPRC siRNAs in HeLa-S3-FlpIn control and BCDIN3D-KO cells. ( B ) Ratio of LRPPRC over β-Tubulin normalized to control in quantitative LI-COR western blots of HeLa-S3-FlpIn control and BCDIN3D-KO whole-cell extracts (mean ± SD, n = 3 independent biological replicates, * P value = 0.03 for LRPPRC in a paired ratio t test). ( C ) Polysome lysates from HeLa-S3-FlpIn control and BCDIN3D-KO cells were fractionated on a 7–50% sucrose gradient and shown are from top to bottom: the real-time recording of OD 254 ; western blots with the indicated antibodies of 20 µL of each fraction (asterisk indicates a non-specific band detected by the BCDIN3D antibody); RTqPCR analysis of LRPPRC, PGK1 and B2M mRNA from each fraction of the same polysome fractionation (shown is mean from two technical replicates).

Techniques: Immunoprecipitation, Western Blot, Staining, Comparison, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Recombinant, Northern Blot

Journal: EMBO Reports

Article Title: ChemRAP uncovers specific mRNA translation regulation via RNA 5′ phospho-methylation

doi: 10.1038/s44319-024-00059-z

Figure Lengend Snippet: ( A ) Representative quantitative LI-COR western blots with antibodies against LRPPRC (green) and β-Tubulin (red) of whole-cell extracts collected after 48 h of reverse transfection of siNC and siLRPPRC siRNAs in HeLa-S3-FlpIn control and BCDIN3D-KO cells. ( B ) Ratio of LRPPRC over β-Tubulin normalized to control in quantitative LI-COR western blots of HeLa-S3-FlpIn control and BCDIN3D-KO whole-cell extracts (mean ± SD, n = 3 independent biological replicates, * P value = 0.03 for LRPPRC in a paired ratio t test). ( C ) Polysome lysates from HeLa-S3-FlpIn control and BCDIN3D-KO cells were fractionated on a 7–50% sucrose gradient and shown are from top to bottom: the real-time recording of OD 254 ; western blots with the indicated antibodies of 20 µL of each fraction (asterisk indicates a non-specific band detected by the BCDIN3D antibody); RTqPCR analysis of LRPPRC, PGK1 and B2M mRNA from each fraction of the same polysome fractionation (shown is mean from two technical replicates). Normalization was done over the average Ct of each mRNA, which did not show significant differences in Control and BCDIN3D-KO cells. See also Appendix Fig. for more details. ( D ) Harringtonine-treated Ribo-seq data for the LRPPRC and PGK1 mRNAs translation initiation sites are shown on the UCSC genome browser (hg38). ( E ) Schematic of the LRPPRC-5′UTR-GFP reporter. ( F ) Flow cytometry analysis of GFP intensity in HeLa-S3-FlpIn control and BCDIN3D-KO cells with a single copy of the LRPPRC-5′UTR-GFP reporter at the FRT locus. Shown are also HeLa-S3-FlpIn cells without reporter (− in yellow) as a negative control. Left: Histogram distribution of GFP intensity (Arbitrary Units) from ~30,000 cells per sample. Right: violin plot of GFP intensity (Arbitrary Units) from ~30,000 single cells per sample. (**** P value < 0.0001 in one-way ordinary ANOVA with Tukey’s multiple comparisons test, only the result of the control/BCDIN3D-KO pair is shown). ( G ) Representative quantitative LI-COR western blots of LRPPRC, PGK1, β-Tubulin, EPRS and MARS distribution upon mitochondrial enrichment in HeLa-S3-FlpIn control and BCDIN3D-KO cells. Shown are also BCDIN3D and two mitochondrial markers (ATPIF1 and HSP60). Sup. stands for supernatant, and Mito. stands for mitochondria-enriched fraction. ( H ) Ratio of LRPPRC, PGK1, EPRS, and MARS over β-Tubulin normalized to the control mitochondria-enriched fraction of quantitative LI-COR western blots (mean ± SD, n = 3–4 independent biological repeats, * P value = 0.047 for LRPPRC and * P value = 0.057 for MARS in a multiple paired ratio t test). Sup. stands for supernatant, and Mito. stands for mitochondria-enriched fraction. ( I ) Left: Representative images of HeLa-FlpIn siNC and siBCDIN3D cells having a single copy of the LRPPRC-5′UTR-GFP reporter at the FRT locus and stained for 15 min with Mitotracker-Red. Right: Raw ImageJ profile analysis of the line shown on the siBCDIN3D merged image for each channel. .

Article Snippet: Figure 6 BCDIN3D regulates LRPPRC translation. ( A ) Representative quantitative LI-COR western blots with antibodies against LRPPRC (green) and β-Tubulin (red) of whole-cell extracts collected after 48 h of reverse transfection of siNC and siLRPPRC siRNAs in HeLa-S3-FlpIn control and BCDIN3D-KO cells. ( B ) Ratio of LRPPRC over β-Tubulin normalized to control in quantitative LI-COR western blots of HeLa-S3-FlpIn control and BCDIN3D-KO whole-cell extracts (mean ± SD, n = 3 independent biological replicates, * P value = 0.03 for LRPPRC in a paired ratio t test). ( C ) Polysome lysates from HeLa-S3-FlpIn control and BCDIN3D-KO cells were fractionated on a 7–50% sucrose gradient and shown are from top to bottom: the real-time recording of OD 254 ; western blots with the indicated antibodies of 20 µL of each fraction (asterisk indicates a non-specific band detected by the BCDIN3D antibody); RTqPCR analysis of LRPPRC, PGK1 and B2M mRNA from each fraction of the same polysome fractionation (shown is mean from two technical replicates).

Techniques: Western Blot, Transfection, Fractionation, Flow Cytometry, Negative Control, Staining