Journal: bioRxiv

Article Title: Ectopic FVIII expression and misfolding in hepatocytes as a potential cause of human hepatocellular carcinoma

doi: 10.1101/2021.01.19.427220

Figure Lengend Snippet: CHO cells that express BDD or N6 were treated with either normal media or glucose-free media containing 10 mM 2DG and 20 mM azide (2DG+NaN 3 ). After 2 hr, cells were harvested or allowed to recover in complete media for the indicated times, or complete media with 10 mg/ml CHX. Cell lysates were filtered through nitrocellulose (left) or cellulose acetate (right) membranes and probed with FVIII or β-actin antibodies. Analysis of β-actin is from BDD expressing CHO cells. B) Reversible retention and secretion of active FVIII in CHO cells. [ 35 S] met/cys pulse-chase and aPTT analysis of BDD and N6 expressed in CHO cells. CHO cells were treated in parallel as in Panel A. CHO cells were pulse-labeled for 20 min and then chased for 20 min with media containing excess unlabeled met/cys to complete synthesis of nascent chains (lane 1) before being treated with either normal media (lanes 2-3) or glucose-free media containing 10 mM 2DG and 20 mM NaN 3 . After 2 hr, cells were harvested (lane 4) or allowed to recover in complete media for increasing times (lanes 5-8), or complete media with CHX (lane 9). Lysates and media were collected at indicated time points for FVIII immunoprecipitation and reducing SDS-PAGE. For FVIII aPPT activity assay of media, cells were treated in parallel, but not pulse-labeled. Lanes: 1: Untreated, 20’ chase; 2: Untreated 120’ chase; 3: Untreated 240’ chase; 4-9: 2DG for 120’; 5: 2-DG + 30’ recovery; 6: 2-DG + 60’ recovery; 7: 2DG + 120’ recovery; 8: 2DG + 240’ recovery; 9: 2DG + 240’ recovery + CHX.

Article Snippet: Mouse anti-human FVIII was from Green Mountain Antibodies (Burlington, VT); Mouse anti-CHOP was from Santa Cruz Biotechnology (Santa Cruz, CA); Rabbit anti-CRELD2 and mouse anti-hepcidin antibodies were from Proteintech (Rosemont, IL) and Novus Biologicals (Burlington, Vt), respectively.

Techniques: Expressing, Pulse Chase, Labeling, Immunoprecipitation, SDS Page, Activity Assay

Journal: bioRxiv

Article Title: Ectopic FVIII expression and misfolding in hepatocytes as a potential cause of human hepatocellular carcinoma

doi: 10.1101/2021.01.19.427220

Figure Lengend Snippet: Epitope-tagging of the endogenous murine BIP/GRP78/Hspa5 locus demonstrates misfolding of BDD in vivo . A: A diagram illustrating location of insertion of 3x FLAG tag to C-terminal region of BiP; B: Western blot demonstrating similar changes in hepatic levels of wild-type BiP (BiP) and BiP-FLAG in mice heterozygous for BiP-Flag allele (10 days after activation of BiP-FLAG expression in the liver) in response to treatment with tunicamycin for 24 h. C. Co-immunoprecipitation (IP) of BDD (lanes 13 &14) and N6 (lanes 17 & 18) from liver lysates of BDD or N6 vector-injected BiP-Flag homozygous mice ( BiP-Flag +/+ ) with anti-FLAG magnetic beads. Direct FVIII IP using anti-FVIII agarose beads demonstrates the relative levels of BDD and N6 in the liver samples (lanes 21-24).

Article Snippet: Mouse anti-human FVIII was from Green Mountain Antibodies (Burlington, VT); Mouse anti-CHOP was from Santa Cruz Biotechnology (Santa Cruz, CA); Rabbit anti-CRELD2 and mouse anti-hepcidin antibodies were from Proteintech (Rosemont, IL) and Novus Biologicals (Burlington, Vt), respectively.

Techniques: In Vivo, FLAG-tag, Western Blot, Activation Assay, Expressing, Immunoprecipitation, Plasmid Preparation, Injection, Magnetic Beads