Journal: iScience

Article Title: A seven-transmembrane protein-TM7SF3, resides in nuclear speckles and regulates alternative splicing

doi: 10.1016/j.isci.2022.105270

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-H2B , Millipore , Cat#07371 RRID: AB_310561.

Techniques: Recombinant, Blocking Assay, SYBR Green Assay, Software

Journal: Molecular cell

Article Title: Nucleus-translocated ACSS2 promotes the gene transcription for lysosomal biogenesis and autophagy

doi: 10.1016/j.molcel.2017.04.026

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal antibody anti-acetyl-histone H2B , EMD Millipore , Cat#07-373; RRID:AB_211814 4.

Techniques: Transduction, Recombinant, Transfection, SYBR Green Assay, Chromatin Immunoprecipitation, Fractionation, Double Knockout, shRNA, Sequencing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Software

Journal: The Journal of Biological Chemistry

Article Title: Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Aggregation Causes Mitochondrial Dysfunction during Oxidative Stress-induced Cell Death

doi: 10.1074/jbc.M116.759084

Figure Lengend Snippet: Formation of GAPDH aggregates occurs at mitochondria. A , confirmation of mitochondrial fractionation using CIV as a mitochondrial marker, H2B as a nuclear marker, and TPI as a cytosolic marker. GAPDH was present in the mitochondrial fractions ( left panel ) from SH-SY5Y cells, which were subjected to non-reduced Western blotting ( WB ) for GAPDH ( right panel ). B , images of cell immunofluorescence of GAPDH ( green ) and MitoTracker Red ( red ) are shown. GAPDH-positive punctate signals in cells treated with NOC18 partially colocalize with MitoTracker Red. Scale bar , 5 μm. C , Western blotting for GAPDH and Coomassie Brilliant Blue (CBB) staining of mitochondrial fractions in the absence (non-reduced) or presence (reduced) of 100 m m DTT. The graphs represent quantitative results of Western blotting analysis regarding GAPDH oligomers ( Oligo .) and monomers ( Mono .). Data are mean ± S.D. ( n = 3). *, p < 0.05, relative to NOC18(−), Student's t test.

Article Snippet: Antibodies were as follows: mouse monoclonal anti-GAPDH (MAB374) and rabbit polyclonal anti-histone H2B (catalog No. 07-371) from Millipore; rabbit polyclonal anti-AIF (AF1475) from R&D Systems; mouse monoclonal anti-cyt c (catalog No. 556433) from BD Biosciences; rabbit polyclonal anti-Myc (A-14, sc-789), rabbit polyclonal anti-CHOP (F-168, sc-575), and rabbit polyclonal anti-His (Omni-probe M-21, sc-499) from Santa Cruz Biotechnology; mouse monoclonal anti-CIV (1D6E1A8) from Invitrogen; monoclonal anti-KDEL (GRP78) (10C3) from Stressgen; monoclonal anti-β-actin (AC15) from Sigma; mouse monoclonal anti-LC3 (2G6) from NanoTools; and polyclonal anti-triosephosphate isomerase (TPI) prepared in house ( ).

Techniques: Fractionation, Marker, Western Blot, Immunofluorescence, Staining

Journal: The Journal of Biological Chemistry

Article Title: Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Aggregation Causes Mitochondrial Dysfunction during Oxidative Stress-induced Cell Death

doi: 10.1074/jbc.M116.759084

Figure Lengend Snippet: GAPDH aggregate-mediated mitochondrial dysfunction participates in release of cyt c and nuclear translocation of AIF. A , release of cyt c into the cytosol of SH-SY5Y cells was induced by treatment with NOC18 for 48 h, as evaluated by cytosolic fractionation and Western blotting ( WB , left panel ). The graph presents values calculated as the ratio of cyt c band intensity relative to TPI band intensity. Data are mean ± S.D. ( n = 3). **, p < 0.01, relative to control, Student's t test. B , effects of the DOX-induced overexpression of WT- and C152A-GAPDH on cyt c release after NOC18 treatment. Data are mean ± S.D. ( n = 3). *, p < 0.05, relative to DOX(−), Student's t test. C , immunofluorescence of cyt c ( green ) and MitoTracker Red ( red ) in DOX-inducible WT- or C152A-GAPDH-overexpressing SH-SY5Y cells treated with NOC18 for 48 h. Scale bar , 10 μm. D , nuclear translocation of AIF was assessed by Western blotting of nuclear fractions. Values were calculated as the ratio of AIF band intensity to H2B band intensity. Data are mean ± S.D. ( n = 3). **, p < 0.01, relative to vehicle treatment, Student's t test. E , effects of overexpression of WT- and C152A-GAPDH on nuclear translocation of AIF induced by NOC18 treatment. Data are mean ± S.D. ( n = 3). *, p < 0.05, **, p < 0.01, relative to DOX(−), Student's t test. F , immunofluorescence for AIF ( green ) and MitoTracker Red ( red ) in DOX-inducible WT- or C152A-GAPDH-overexpressing SH-SY5Y cells. Nuclei were stained with DAPI ( blue ). Scale bar , 10 μm. G and H , effect of CsA on NOC18-induced release of cyt c and nuclear translocation of AIF, respectively. Data are mean ± S.D. ( n = 3). *, p < 0.05, **, p < 0.01, relative to CsA(−), Student's t test.

Article Snippet: Antibodies were as follows: mouse monoclonal anti-GAPDH (MAB374) and rabbit polyclonal anti-histone H2B (catalog No. 07-371) from Millipore; rabbit polyclonal anti-AIF (AF1475) from R&D Systems; mouse monoclonal anti-cyt c (catalog No. 556433) from BD Biosciences; rabbit polyclonal anti-Myc (A-14, sc-789), rabbit polyclonal anti-CHOP (F-168, sc-575), and rabbit polyclonal anti-His (Omni-probe M-21, sc-499) from Santa Cruz Biotechnology; mouse monoclonal anti-CIV (1D6E1A8) from Invitrogen; monoclonal anti-KDEL (GRP78) (10C3) from Stressgen; monoclonal anti-β-actin (AC15) from Sigma; mouse monoclonal anti-LC3 (2G6) from NanoTools; and polyclonal anti-triosephosphate isomerase (TPI) prepared in house ( ).

Techniques: Translocation Assay, Fractionation, Western Blot, Over Expression, Immunofluorescence, Staining