representative western blot  (Proteintech)

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

Journal: Nature Communications

Article Title: NUP62 localizes to ALS/FTLD pathological assemblies and contributes to TDP-43 insolubility

doi: 10.1038/s41467-022-31098-6

Figure Lengend Snippet: a HEK293 cells were co-transfected with mRuby-NUP62 and eGFP-TDP-43 (wild type). The cells were observed through live-scan confocal microscopy starting 3 h after transfection and images were obtained every 5 min over the course of 15 h. Two populations of cytoplasmic mRuby-NUP62 condensates were observed: reversible or irreversible. Reversible structures exhibit more dynamic activity and appear circular (see arrow). Irreversible structures appear less mobile or more static and have an angular structure (see asterisks). b Schematic depicting characteristics of cytoplasmic mRuby-NUP62 structures is shown at bottom. Representative still images were obtained from the 6–10 h time points of the imaging session. c Quantification of NUP62 area in confocal microscopy images obtained during live imaging (5–15 h timepoints) described in Fig. 5A, B. Irreversible condensates were significantly larger than reversible structures. The size of reversible granules was determined at times point immediately prior to dissipation. Irreversible granule area was calculated at final time point collected during live imaging session. n = 50 (reversible), 20 (irreversible) NUP62 + granules. Statistical significance was determined by two-tailed, unpaired student’s t-test. Data are shown as mean + /- SEM. d The percentage of reversible and irreversible mRuby-NUP62 granules containing eGFP-TDP-43 were calculated for each frame taken throughout the duration of living imaging session (5–15 h timepoints) described in Fig. 5a, b. A greater percentage of irreversible mRuby-NUP62 condensates contained eGFP-TDP43. n = 12 frames per group. Statistical significance was determined by two-tailed, unpaired student’s t-test. Data are shown as mean + /- SEM. e mRuby-NUP62 condensates were characterized for circularity score at the final time point of live image session (5–15 h timepoints) described in Fig. 5a, b. Irreversible mRuby-NUP62 + eGFP-TDP-43 + condensates ( n = 26 condensates) had a significantly reduced circularity score relative to eGFP-TDP-43 - ( n = 17 condensates) and reversible mRuby-NUP62 + eGFP-TDP-43 + condensates ( n = 12 condensates). Statistically differences were calculated by one-way ANOVA with Tukey post hoc analysis. Data are shown as mean + /- SEM. f Representative FRAP analysis images of nuclear eGFP-TDP-43 (reference solubility control) and cytoplasmic eGFP-TDP-43 and mRuby-NUP62 condensates. g Quantification of FRAP analysis shows reduced fluorescence signal recovery in cytoplasmic eGFP-TDP-43 and mRuby-NUP62 condensates relative to nuclear eGFP-TDP-43 control. Data are shown as mean + /- SD. h HEK293 cells were transfected with indicated plasmids for 24 h. Soluble and insoluble biochemical fractionation was then conducted, and Western blot analysis was performed to evaluate TDP-43 and GAPDH (protein loading control). mRuby-NUP62 promotes the formation of increased insoluble TDP-43. Representative western blot image is shown. i HEK293 cells were transfected with mRuby-NUP62 and eGFP-TDP-43 (WT or ΔNLS) for 24 h. Samples were then immunoprecipitated by ChromoTek GFP-Trap Magnetic Agarose affinity beads. Samples were then immunoblotted for NUP62 and TDP-43. * p ≤ 0.05; ** p ≤ 0.01; **** p ≤ 0.0001 vs control. Scale bar: 10 µm.

Article Snippet: Representative western blot image is shown. i HEK293 cells were transfected with mRuby-NUP62 and eGFP-TDP-43 (WT or ΔNLS) for 24 h. Samples were then immunoprecipitated by ChromoTek GFP-Trap Magnetic Agarose affinity beads.

Techniques: Transfection, Confocal Microscopy, Activity Assay, Imaging, Two Tailed Test, Solubility, Control, Fluorescence, Fractionation, Western Blot, Immunoprecipitation