human apob48  (Addgene inc)

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: Hepatocyte-specific Pdia1 deletion induces severe hypolipidemia and hepatic steatosis with a blockade of hepatic TG export. A. Schematic shows floxed exons 1 and 2 of the Pdia1 allele ( Pdia1 f/f ). Generation of hepatocyte-specific PDIA1-ablated mice ( Pdia1 -LKO) was produced using Ad-CMV-Cre or AAV8-TBG-Cre. B. Transduction of Pdia1 f/f with AAV8-TBG-Cre eliminated Pida1 mRNA in the liver but did not affect the mRNA levels of other PDI protein family members. C. PDIA1 was absent in the livers of Pdia1 -LKO accompanied with upregulation of PDIA4. D. Plasma levels of total cholesterol and TG were both greatly reduced in Pdia1 -LKO mice (n = 5). E and F. APOB100, APOB48 and APOE were nearly absent in plasma of Pdia1 -LKO mice, with a reduced level of ApoA1 but plasma albumin levels were not changed. Each lane represents a sample from individual mouse. G. Liver images and H/E-stained liver sections demonstrate severe liver fat accumulation in Pdia1 -LKO mice. Scale bar: 200 μm. H. Hepatic TG content was markedly increased in Pdia1 -LKO mice (n = 5). I. Dramatic decrease in hepatic VLDL-TG secretion in Pdia1 -LKO mice (n = 3). ∗∗P < 0.01.

Article Snippet: Expression vector encoding human APOB48 was from Addgene (138334; Watertown, MA).

Techniques: Produced, Transduction, Clinical Proteomics, Staining

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: Expression vector encoding human APOB48 was from Addgene (138334; Watertown, MA).

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

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: Wild type PDIA1 (PDI) and catalytically inactive PDIA1 (PDImt) directly interact with the peptide region between APOB27 and APOB48, respectively . A. PDI and PDImt interact with APOB48 in transfected COS-7 cells. COS-7 cells were transfected with human APOB48 (hAPOB48), C-terminal-FLAG-tagged PDI (PDI-f) or PDImt (PDImt-f) expression vectors as indicated. The transfected cells were harvested 30 h post-transfection and subjected to FLAG-immnunoprecipitation (IP) analysis using M2 anti-FLAG magnetic beads. Co-IP of hAPOB48 with PDI-f (lanes 10 & 11) or PDImt-f (lanes 12 & 13) indicate their direct interactions. B. Neither APOB17 nor APOB27 interact with PDI or PDImt. COS-7 cells were transfected PDI-f or or PDImt-f expression vectors in the presence of Ad-hAPOB15 (hAPOB15) or Ad-hAPOB27. FLAG-IP assays were performed on the DNA transfected COS-7 cells at 30 h post-transfection. No hAPOB15 nor hAPOB27 were pulled down with PDI-f or PDImt-f. C. PDIA1 is not required for secretion of APOB17 and APOB27. Hepatocytes isolated from Pdia1 f/f and Pdia1 -LKO mice were transduced with Ad-GFP (GFP), Ad-hAPOB15 (B15), or Ad-hAPOB27 (B27). At 48 h post-transduction, hepatocytes, and conditioned media (20 h incubation time) were harvested. Cellular and secreted human ApoB15 and apoB27 were immunoprecipitated with rabbit anti-human ApoB followed by immunoblot analysis using goat anti-human ApoB.

Article Snippet: Expression vector encoding human APOB48 was from Addgene (138334; Watertown, MA).

Techniques: Transfection, Expressing, Magnetic Beads, Co-Immunoprecipitation Assay, Isolation, Transduction, Incubation, Immunoprecipitation, Western Blot