Journal: Biochemical Journal

Article Title: p32 protein levels are integral to mitochondrial and endoplasmic reticulum morphology, cell metabolism and survival

doi: 10.1042/BJ20121829

Figure Lengend Snippet: HeLa cells were pre-treated with p32-specific siRNA (60 nM) or control siRNA for 48 h. ( A ) Cells were stained with MitoTrackerRed (mitochondria), immunostained with anti-calnexin antibodies (ER), and then stained with DAPI (nuclei). The Merge panels overlay MitoTrackerRed, calnexin and DAPI staining. ( B ) Cells were stained with MitoTrackerRed (mitochondria), immunostained with anti-α-tubulin antibodies, then stained with DAPI (nuclei). The merge panels overlay MitoTrackerRed, α-tubulin (microtubules) and DAPI staining. Scale bar is 10 μm. ( C ) siRNA-treated cells were immediately subject to the sample preparation and TEM analysis. TEM images show ER with arrowheads, associated ribosomes with hollow arrowheads and mitochondrion denoted M. Panels b and e are high magnification (×3) images of the boxed areas in panels a and d respectively. Results are representative of three independent experiments. ( D ) Control and p32 siRNA-treated HeLa cells were subjected to high pressure freezing, immunogold labelling and cryo-TEM analysis. Individual mitochondrion and ER were highlighted by Mito and arrows respectively. Black arrowheads indicate ER–mitochondria contact points, and white arrowheads indicate ER not in contact with mitochondria. Results were representative images taken from a pool of 25 images for each condition.

Article Snippet: The primary antibodies used and their dilutions were as follows: mouse monoclonal anti-(p32 N m -terminus) antibody (1:100 dilution; Abcam); mouse monoclonal anti-c-Myc antibody (1:100 dilution, sc-40; Santa Cruz Biotechnology); rabbit anti-calnexin antibody (1:100 dilution, C4731; Sigma–Aldrich); and mouse monoclonal anti-α-tubulin (1:100 dilution, sc-5286, Santa Cruz Biotechnology).

Techniques: Staining, Sample Prep

Journal: Journal of Lipid Research

Article Title: Role of N -glycosylation of human lysosomal phospholipase A2 for the formation of catalytically active enzyme

doi: 10.1194/jlr.M041640

Figure Lengend Snippet: Subcellular fractionation of WT and mutated hLPLA2 gene transfectants by differential centrifugation. All manipulations in the subcellular fractionation were carried out at 4°C. One milliliter of cell homogenate (1 mg protein/ml) obtained from WT, N1A, or Nt gene transfectants was centrifuged for 10 min at 600 g. The resultant supernatant and pellet were collected as post nuclear supernatant (PNS) and nuclear fraction, respectively. The PNS was centrifuged for 10 min at 15,000 g. The resultant pellets were collected as crude mitochondrial fraction (MIT). The supernatant obtained at 15,000 g was centrifuged for 1.5 h at 160,000 g. The resultant supernatant and pellet were collected as soluble fraction (SOL) and microsomal fraction (MIC), respectively. The pellet of MIT and MIC was dispersed with 1 ml of 0.25 M sucrose, 10 mM HEPES (pH 7.4), and 1 mM EDTA. Twenty microliters of the cell homogenate (HOM), PNS, MIT, and MIC suspensions, and SOL was used to assure the distribution of LPLA2 in the cell. SDS polyacrylamide gel electrophoresis and Western blotting were carried out as described in the Materials and Methods. In (A), distribution of WT in the cell was examined by treatment of the membrane with anti-hLPLA2 antibody (LP) or anti-lamp1 antibody (LM). In (B), N1A and Nt were found in MIC. The left and middle membranes were treated with anti-hLPLA2 antibody. The right membrane was treated with anti-calnexin antibody.

Article Snippet: Reagents 1,2-Dioleoyl- sn -glycero-3-phosphocholine (DOPC), sulfatide, N -acetylsphingosine (NAS), and N -oleoyl-sphingosine were obtained from Avanti Polar Lipids Corp. (Alabaster, AL); high performance thin layer chromatography silica gel plates, 10 × 20 cm, were from Merck (Darmstadt, Germany); N -glycosidase F (PNGase F) and POD immunostain set were from Wako (Tokyo, Japan); anti-rabbit IgG goat polyclonal antibody, HRP-conjugate, was from MP Biomedicals (Solon, OH); anti-lamp1 mouse monoclonal antibody and anti-calnexin rabbit polyclonal antibody were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Fractionation, Centrifugation, Polyacrylamide Gel Electrophoresis, Western Blot, Membrane

Journal: Cell Death & Disease

Article Title: BAX inhibitor-1-associated V-ATPase glycosylation enhances collagen degradation in pulmonary fibrosis

doi: 10.1038/cddis.2014.86

Figure Lengend Snippet: Lysosomal V-ATPase is highly expressed in BI-1 cells. ( a ) Neo and BI-1 cells were cultured in serum-free medium with or without 5 ng/ml TGF- β 1 for 0, 12, 24, 36, or 48 h. Immunoblotting was performed with antibodies against the V-ATPase V0a1 subunit or β -actin. Ratio of glycosylated to unglycosylated V-ATPase was quantified as shown in the right panel. * P <0.05, significantly different from Neo cells during each period. ( b ) Neo and BI-1 cells were cultured in serum-free medium with or without 5 ng/ml TGF- β 1 for 0, 24, or 48 h. After the lysosome and ER fractions were isolated, immunoblotting was performed using anti-V-ATPase V0a1 subunits, LAMP-1, or IP3R antibodies. ( c ) Neo and BI-1 cells were cultured in serum-free medium with or without 5 ng/ml TGF- β 1 for 48 h. Immunostaining with anti-V-ATPase, calnexin, or LAMP-1 antibodies was performed, and the images were captured under a fluorescent microscope

Article Snippet: Anti-mouse collagen antibody for immunoblotting, anti-mouse monoclonal V-ATPase antibody for immunostaining, β -actin antibody, anti-rabbit polyclonal calnexin antibody, anti-rat monoclonal LAMP-1 antibody, anti-rat monoclonal GRP78 antibody, anti-rabbit polyclonal CHOP antibody, and anti-mouse monoclonal SMA antibody were supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Cell Culture, Western Blot, Isolation, Immunostaining, Microscopy

Journal: Cell Death & Disease

Article Title: BAX inhibitor-1-associated V-ATPase glycosylation enhances collagen degradation in pulmonary fibrosis

doi: 10.1038/cddis.2014.86

Figure Lengend Snippet: BI-1 enhances N-glycosylation activity in the ER during TGF- β 1 treatment. ( a ) Neo and BI-1 cells were cultured with 5 ng/ml TGF- β 1 for 0, 24, or 48 h. O-glynase, Endo-H, or PNGase-F was added to the extracts followed by 30-min incubation. Immunoblotting was then performed with anti-V-ATPase antibody. ( b ) V-ATPase activity in the lysosome fractions isolated from 5 ng/ml TGF- β 1-treated Neo or BI-1 cells in the presence or absence of PNGase-F was measured and quantified. * P <0.05, significantly different from TGF- β 1-treated BI-1 cells. ( c ) Purified collagens from rat tails were incubated with lysosome fractions from TGF- β 1-treated or non-treated Neo or BI-1 cells in the presence or absence of PNGase-F. Coomassie staining was performed as described in the Materials and Methods. * α 1 or α 2 collagen. ( d ) Neo and BI-1 cells were cultured in serum-free medium with 5 ng/ml TGF- β 1 for 0, 12, 24, 36, or 48 h. After isolating a pure ER fraction, α -glucosidase, ER-resident mannosidase or UDP glucuronsyltransferase activity was analyzed as described in the Materials and Methods. * P <0.05, significantly different from Neo cells at each time point. ( e ) Neo and BI-1 cells were cultured in serum-free medium with 5 ng/ml TGF- β 1 for 0, 12, 24, 36, or 48 h, and immunoblotting was performed with anti-calreticulin or calnexin antibodies. β -Actin antibody staining of the same loading was used as a control. ( f ) Neo and BI-1 cells were cultured in serum-free medium with 5 ng/ml TGF- β 1 for 0, 24, or 48 h. Lysates were subjected to immunoprecipitation and immunoblotting with anti-V-ATPase or calnexin antibody

Article Snippet: Anti-mouse collagen antibody for immunoblotting, anti-mouse monoclonal V-ATPase antibody for immunostaining, β -actin antibody, anti-rabbit polyclonal calnexin antibody, anti-rat monoclonal LAMP-1 antibody, anti-rat monoclonal GRP78 antibody, anti-rabbit polyclonal CHOP antibody, and anti-mouse monoclonal SMA antibody were supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Glycoproteomics, Activity Assay, Cell Culture, Incubation, Western Blot, Isolation, Purification, Staining, Control, Immunoprecipitation

Journal: Cell Death & Disease

Article Title: BAX inhibitor-1-associated V-ATPase glycosylation enhances collagen degradation in pulmonary fibrosis

doi: 10.1038/cddis.2014.86

Figure Lengend Snippet: BI-1 regulates lung fibrosis through enhanced ER folding capacitance-associated lysosomal V-ATPase glycosylation. BI-1 +/+ and BI-1 −/− mice were treated by intratracheal injection of bleomycin (0.5 U/kg) for the indicated number of weeks. ( a ) Electron microscopy was performed as described in the Materials and Methods. ( b ) After isolation of lysosomes from lung tissue, cathepsin B and K enzyme activities were analyzed. ( c ) To assess lysosomal V-ATPase activity, 6 μ M acridine orange solution was added to lysosomal membranes from the lung tissues of bleomycin-treated or non-treated mice for the indicated periods. Intra-vesicular H + uptake was initiated by the addition of Mg-ATP. Lysosomal V-ATPase activity was analyzed as described in the Materials and Methods. * P <0.05, significantly different from ATP-exposed BI-1 +/+ control mice. ( d ) β -Galactosidase and α -mannosidase activities were measured in pure lysosomal extracts. * P <0.05, significantly different from ATP-exposed BI-1 +/+ control mice. ( e ) Immunoblotting of lung lysates from BI-1 +/+ and BI-1 −/− mice treated with bleomycin for 0, 2, or 4 weeks was performed using antibodies against V-ATPase, calnexin, and calreticulin. ( f ) Immunoprecipitation and immunoblotting of the lysates were performed using anti-V-ATPase or calnexin antibody

Article Snippet: Anti-mouse collagen antibody for immunoblotting, anti-mouse monoclonal V-ATPase antibody for immunostaining, β -actin antibody, anti-rabbit polyclonal calnexin antibody, anti-rat monoclonal LAMP-1 antibody, anti-rat monoclonal GRP78 antibody, anti-rabbit polyclonal CHOP antibody, and anti-mouse monoclonal SMA antibody were supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Glycoproteomics, Injection, Electron Microscopy, Isolation, Activity Assay, Control, Western Blot, Immunoprecipitation