rabbit polyclonal anti mouse apob  (Millipore)

Journal: Molecular Metabolism

Article Title: Conditional hepatocyte ablation of PDIA1 uncovers indispensable roles in both APOB and MTTP folding to support VLDL secretion

doi: 10.1016/j.molmet.2024.101874

Figure Lengend Snippet: Secretion of APOB48 is completely blocked in Pdia1 -deleted hepatocytes and is rescued by complementary expression of wild type PDIA1 (PDI) or catalytically inactive PDIA1 (PDImt). A . Pulse-chase analysis revealed that Pdia1 -deletion did not affect APOB48 synthesis (lane 5 vs lane 1) but it completely inhibited APOB48 secretion (lanes 12–14 vs lanes 9–11, respectively). B . Complementary expression of PDI or PDImt alone rescued APOB48 secretion. Hepatocytes isolated from Pdia1 f/f and Pdia1 -LKO mice were infected with the indicated adenoviruses at 20 h after plating. At 18 h post-transduction, the hepatocytes were pulse-labeled with 35 S-Met/Cys in the presence of 0.3 mM oleic acid complexed with BSA (OA-BSA) for 3 h. The 35 S-labeled ApoB's and albumin were immunoprecipitated with rabbit polyclonal antibodies against mouse APOB and albumin, respectively. Immunoblotting (IB) demonstrated that no endogenous MTTP was rescued in the Ad-PDI- or Ad-PDImt-infected Pdia1 -LKO hepatocytes. C. Complementary expression of PDI or PDImt alone did not rescue secretion of 3 H-labeled TG by the Pdia1 -LKO hepatocytes, neither did forced expression of MTTP alone. Hepatocytes isolated from Pdia1 f/f and Pdia1 -LKO mice were infected with the indicated adenoviruses. At 18 h p.i., hepatocytes were incubated with DMEM containing 0.3 mM oleic acid-BSA and 3 H-glycerol for 4 h. The 3 H-labeled TG in cells and media were isolated and the 3 H-radioacivity was measured and expressed as DPM/mg cell protein/h. Each bar represents average +/− SD of triplicate wells. ∗, P < 0.05; ∗∗, P < 0.01.

Article Snippet: ApoB and albumin in the cell lysates and chase media were immunoprecipitated using rabbit polyclonal anti-mouse APOB [ , ] and rabbit polyclonal anti-human albumin (Sigma) and the 35 S-labeled APOB and albumin were quantified as described above for 35 S-labeled MTTP.

Techniques: Expressing, Pulse Chase, Isolation, Infection, Transduction, Labeling, Immunoprecipitation, Western Blot, Incubation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Hepatic TM6SF2 Is Required for Lipidation of VLDL in a Pre-Golgi Compartment in Mice and Rats

doi: 10.1016/j.jcmgh.2021.12.008

Figure Lengend Snippet: Generation and characterization of Tm6sf2 -/- rats using CRISPR/Cas9 technology. ( A ) Diagram of CRISPR/Cas9-targeted disruption of rat Tm6sf2 by insertion of T in exon 1 (c.80_81insT). Sequences of the guide RNA (gRNA) binding site, protospacer adjacent motif (PAM), and introns (green) are indicated. ( B ) TM6SF2 mRNA levels in female rat livers (n = 3–5/group, 3–4 wk) were determined using real-time PCR and normalized to levels of cyclophilin B mRNA. ( C ) Immunoblot of liver lysates ( left ) and enterocytes ( right ) from 4- to 5-week-old rats. ( D ) Body weights, liver weights, and hepatic lipids of chow-fed male rats (n = 11–19/group, 3–4 wk) after a 4-hour fast. ( E ) Plasma lipid levels were measured in the same rats as used in panel D ( left ). Plasma samples from WT and Tm6sf2 -/- male rats (4/group, 5–6 wk) were pooled and size-fractionated by fast performance liquid chromatography. Cholesterol and TG levels were measured in each fraction ( right ). ( F ) Plasma (1 μL) was size-fractionated by 4%–15% gradient SDS–polyacrylamide gel electrophoresis. Levels of APOB were determined by immunoblot analysis using a rabbit polyclonal antibody (ab20737). Bars indicate means ± SD. Differences between groups were analyzed using the Student unpaired 2-tailed t test. ∗ P < .05, ∗∗ P < .01, and ∗∗∗ P < .001, compared with the WT group. All experiments were repeated twice, and results were similar. C, colon; CANX, calnexin; D, duodenum; FN1, fibronectin; HDL, high-density lipoprotein; I, ileum; J, jejunum.

Article Snippet: Co-immunoprecipitation of TM6SF2 with ApoB and ACSL5. ( A ) Hepatic lysates from mice expressing adeno-associated virus (AAV)–human V5-tagged TM6SF2 (hTM6SF2-V5) or AAV-luciferase were precleaned with A/G agarose beads and then (500 μg, input) was incubated with anti-V5 agarose beads ( right ) or with rabbit anti-mouse ApoB polyclonal antibody (Abcam Cambridge MA) at 4°C overnight.

Techniques: CRISPR, Binding Assay, Real-time Polymerase Chain Reaction, Western Blot, Liquid Chromatography, Polyacrylamide Gel Electrophoresis

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Hepatic TM6SF2 Is Required for Lipidation of VLDL in a Pre-Golgi Compartment in Mice and Rats

doi: 10.1016/j.jcmgh.2021.12.008

Figure Lengend Snippet: Co-immunoprecipitation of TM6SF2 with ApoB and ACSL5. ( A ) Hepatic lysates from mice expressing adeno-associated virus (AAV)–human V5-tagged TM6SF2 (hTM6SF2-V5) or AAV-luciferase were precleaned with A/G agarose beads and then (500 μg, input) was incubated with anti-V5 agarose beads ( right ) or with rabbit anti-mouse ApoB polyclonal antibody (Abcam Cambridge MA) at 4°C overnight. Input (10%) and IP samples were size-fractionated using SDS–polyacrylamide gel electrophoresis (4%–12%) and immunoblotting was performed as described in the Methods section. ( B ) Mouse intestinal epithelial cells were incubated ± PFA (1%) and then quenched with 1.25 mol/L glycine. Homogenates from PFA-treated cells (PFA-Input) were precleaned with A/G agarose beads and then 500 μg was incubated with mouse anti-mouse TM6SF2 mAb (8B3) ( left ) or rabbit anti-mouse ACSL5 (25D12) ( right ). ( C ) Co-immunoprecipitation of TM6SF2 with ApoB and ACSL5 from rat intestinal epithelial cells. Samples were handled as described in panel A except cells were homogenized in lysis buffer (1% digitonin, 5 mmol/L EDTA, 5 mmol/L ethylene glycol-bis(β-aminoethyl ether)- N,N,N′,N′ -tetraacetic acid plus PI) and a total of 500 μg of the lysate (input) was incubated with rabbit anti-rat TM6SF2 antibody (505E (25 μg) at 4°C overnight. The mixture then was incubated with Dynabead protein G at 4°C for 2 hours. Samples were collected as described in the Methods section. Fraction (10%) were subjected to immunoblot analysis. ∗Nonspecific band. All experiments were repeated at least once, and the results were similar. CANX, calnexin.

Article Snippet: Co-immunoprecipitation of TM6SF2 with ApoB and ACSL5. ( A ) Hepatic lysates from mice expressing adeno-associated virus (AAV)–human V5-tagged TM6SF2 (hTM6SF2-V5) or AAV-luciferase were precleaned with A/G agarose beads and then (500 μg, input) was incubated with anti-V5 agarose beads ( right ) or with rabbit anti-mouse ApoB polyclonal antibody (Abcam Cambridge MA) at 4°C overnight.

Techniques: Immunoprecipitation, Expressing, Luciferase, Incubation, Polyacrylamide Gel Electrophoresis, Western Blot, Lysis