Journal: The Journal of biological chemistry

Article Title: The prespore vesicles of Dictyostelium discoideum. Purification, characterization, and developmental regulation.

doi: 10.1074/jbc.274.50.35823

Figure Lengend Snippet: FIG. 8. Triton X-114 fractionation of aqueous versus mem- brane-bound proteins from purified PSVs. A, PSV proteins from band 3 of the Percoll gradient were fractionated into aqueous and detergent phases, separated on a 7–15% SDS-polyacrylamide gel, and stained with Coomassie Brilliant Blue (lanes 1–3). Positions of molec- ular weight markers are shown on the left. B, Triton X-114-extracted proteins were separated on 10% SDS-PAGE minigels (Bio-Rad) and transferred to nitrocellulose. The blot was probed with the mAb MUD102, which recognizes the soluble protein, PsB. T, total; A, aque- ous phase; D, detergent phase.

Article Snippet: B, Triton X-114-extracted proteins were separated on 10% SDS-PAGE minigels (Bio-Rad) and transferred to nitrocellulose.

Techniques: Fractionation, Purification, Staining, SDS Page

Journal: Science Advances

Article Title: Modulation of pre-mRNA structure by hnRNP proteins regulates alternative splicing of MALT1

doi: 10.1126/sciadv.abp9153

Figure Lengend Snippet: ( A ) MALT1 A and B protein isoforms differ depending on inclusion or exclusion of exon7, which encodes for an 11–amino acid TRAF6 binding domain that regulates downstream function. ( B ) Quantification of endogenous MALT1 transcripts upon KD of hnRNP U, hnRNP L, and hnRNP LL. ( C ) Endogenous protein levels of MALT1A upon KD of hnRNP U and hnRNP L. Asterisk indicates an unspecific band. ( D ) MALT1 minigene constructs that recapitulate splicing regulation of endogenous MALT1 . ( E ) Quantification of MALT1 splicing on minigene constructs upon KD of hnRNP U, hnRNP L, and hnRNP LL. ( F ) EMSA showing that hnRNP U and hnRNP L bind with low nanomolar affinity to the MALT1 minigene RNA. ( G ) EMSAs showing that hnRNP L and hnRNP U compete for binding to the MALT1 minigene RNA. Data are representative for four (B) or three (E) independent experiments. Depicted is the mean ± SD. n = 4 (B) or n = 3 (E). * P < 0.05, ** P < 0.01, and *** P < 0.001; ns, not significant; unpaired Student’s t test. See also fig. S1.

Article Snippet: For minigene assays, 48 hours after siRNA transfection, 2.5 × 10 6 Jurkat T cells were electroporated with 2 μg of minigene constructs using 220 V and 1000 mA (Gene Pulser X, Bio-Rad).

Techniques: Binding Assay, Construct

Journal: Science Advances

Article Title: Modulation of pre-mRNA structure by hnRNP proteins regulates alternative splicing of MALT1

doi: 10.1126/sciadv.abp9153

Figure Lengend Snippet: Binding affinities for minigene RNAs determined from EMSA.

Article Snippet: For minigene assays, 48 hours after siRNA transfection, 2.5 × 10 6 Jurkat T cells were electroporated with 2 μg of minigene constructs using 220 V and 1000 mA (Gene Pulser X, Bio-Rad).

Techniques: Binding Assay

Journal: Science Advances

Article Title: Modulation of pre-mRNA structure by hnRNP proteins regulates alternative splicing of MALT1

doi: 10.1126/sciadv.abp9153

Figure Lengend Snippet: ( A ) SHAPE-derived secondary structure of the MALT1 minigene RNA. Domains 1 and 2 are outlined, with stem-loops (SLs) and splice signals highlighted and annotated. Nonreactive, semireactive, and highly reactive nucleotides are colored white, orange, and red, respectively. ( B ) Binding sites for hnRNP U (blue) and hnRNP L (green) across the MALT1 minigene RNA. ( C and D ) SHAPE-derived secondary structure of variant 1 and variant 2 of MALT1 minigene RNAs, zoomed in to the region that harbors the 5′ and 3′ splice signals flanking exon7. ( E ) Effects and quantification of splicing regulation of exon7 or exon9 (variant 1) upon single or combined KD of hnRNP U and hnRNP L comparing the wild-type (WT) M1, variant 1, and variant 2 minigenes. Data are representative for three independent experiments. Depicted is the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001; ns, not significant; unpaired Student’s t test. See also fig. S2.

Article Snippet: For minigene assays, 48 hours after siRNA transfection, 2.5 × 10 6 Jurkat T cells were electroporated with 2 μg of minigene constructs using 220 V and 1000 mA (Gene Pulser X, Bio-Rad).

Techniques: Derivative Assay, Binding Assay, Variant Assay

Journal: Science Advances

Article Title: Modulation of pre-mRNA structure by hnRNP proteins regulates alternative splicing of MALT1

doi: 10.1126/sciadv.abp9153

Figure Lengend Snippet: ( A ) Raw SHAPE reactivity traces corresponding to SL5, which harbors the 5′ splice site, in the absence of protein (black) and in the presence of hnRNP L (green) and hnRNP U (blue). ( B ) Fluorescence quenching assays with the SL4 RNA hairpin labeled with a fluorescent dye and quencher at the 5′ and 3′ termini show that hnRNP L unwinds, whereas hnRNP U maintains secondary structures of splice signal–containing stem-loops. Errors refer to three biological replicates. ( C ) Minigene splicing assay quantification upon overexpression (1, 2.5, and 5 μg) of various hnRNP U constructs. ( D ) EMSA of hnRNP U RGG domain with MALT1 M1 minigene RNA. ( E ) Minigene splicing assay quantification upon overexpression (5 μg) of various hnRNP L constructs. Data are representative for three (C and E) independent experiments. Depicted is the mean ± SD. n = 3 (C and E). * P < 0.05, ** P < 0.01, and *** P < 0.001; ns, not significant; unpaired Student’s t test. ( F ) EMSA of hnRNP L RRM1 to RRM4 with MALT1 M1 minigene RNA. See also figs. S3 and S4.

Article Snippet: For minigene assays, 48 hours after siRNA transfection, 2.5 × 10 6 Jurkat T cells were electroporated with 2 μg of minigene constructs using 220 V and 1000 mA (Gene Pulser X, Bio-Rad).

Techniques: Fluorescence, Labeling, Splicing Assay, Over Expression, Construct