rat liver fibroblasts  (ATCC)

Journal: bioRxiv

Article Title: N -Glycosylation of MRS2 balances aerobic and anaerobic energy production by reducing rapid mitochondrial Mg 2+ influx in conditions of high glucose or impaired respiratory chain function

doi: 10.1101/2024.07.09.602756

Figure Lengend Snippet: Immunoblot analyses of MRS2 revealed changes following treatment of mitochondrial protein extracts with PNGase F (PNG). Panels A-E show 5 separate gels, where unlabeled lanes display molecular weight (MW) markers. (A) Proteins isolated from mouse liver (MmL) mitoplasts (MTP) ( 1 ) before and ( 2 ) after treatment with PNG. (B) Proteins isolated from mouse liver mitochondria (Mito) and affinity purified with conconavalin A (ConA) beads ( 1 ) before and ( 2 ) after treatment with PNG. (C) Proteins isolated from mouse liver MTP ( 1 ) without PNG treatment and ( 2 ) with PNG treatment and then subsequently affinity purified with Lens culinaris agglutinin (LCA) beads. (D) Proteins isolated from mouse liver AFT024 fibroblast mitochondria ( 1 ) before and ( 2 ) after treatment with PNG and proteins isolated from rat (Rn) liver BRL 3A fibroblast mitochondria ( 3 ) before and ( 4 ) after treatment with PNG. The vertical solid black line in panel D indicates that the two lanes to its right were not immediately adjacent to the molecular weight control lane (MW). (E) Mitochondrial proteins from human fibroblast mitochondria ( 1 ) before and ( 2 ) after treatment with PNG. Overall, 52 and 50 kDa bands were detected in Panels A-C, while 56 and 53 kDa bands were detected in panels D and E.

Article Snippet: BRL 3A rat liver fibroblasts and AFT024 mouse liver fibroblasts were purchased from the American Type Culture Collection (ATCC; Manassas, VA).

Techniques: Western Blot, Molecular Weight, Isolation, Affinity Purification, Control

Journal: bioRxiv

Article Title: N -Glycosylation of MRS2 balances aerobic and anaerobic energy production by reducing rapid mitochondrial Mg 2+ influx in conditions of high glucose or impaired respiratory chain function

doi: 10.1101/2024.07.09.602756

Figure Lengend Snippet: The dotted line corresponds to a ∼ MW of 56 kDa assigned to an N- glycosylated isoform of MRS2 and the dashed line corresponds to a MW of ∼ 53 kDa and is assigned to a nonglycosylated MRS2 isoform. The MW marker band in the left lanes of Panels A and D is assigned a MW of 50 kDa. ( A, B ) Treatment of BRL 3A cells with tunicamycin (TM) for either (A) 24 h at 0.5 µM (B) 24 h at 1.0 µM or (C) 4 h at 2.5 µM or (D) treatment with DON at 100 µM for 24 h, all with companion controls (-). On each of the four separate gels presented in panels A-D, SDS-PAGE immunoblotting with anti-MRS2 revealed a reduction in the 56 kDa/53 kDa intensity ratio. The efficacy of TM varied with both time and concentration while notably the treatment with DON resulted in a near total loss of the N- glycosylated 56 kDa band. The vertical solid black line in panel A indicates that the three lanes shown were not immediately adjacent to the molecular weight marker lane (MW).

Article Snippet: BRL 3A rat liver fibroblasts and AFT024 mouse liver fibroblasts were purchased from the American Type Culture Collection (ATCC; Manassas, VA).

Techniques: Marker, SDS Page, Western Blot, Concentration Assay, Molecular Weight

Journal: bioRxiv

Article Title: N -Glycosylation of MRS2 balances aerobic and anaerobic energy production by reducing rapid mitochondrial Mg 2+ influx in conditions of high glucose or impaired respiratory chain function

doi: 10.1101/2024.07.09.602756

Figure Lengend Snippet: Each of the panels represents western immunoblotting results of separate gels with anti-MRS2. The dotted line corresponds to a ∼ MW of 56 kDa and the dashed line corresponds to a MW of ∼ 53 kDa and is assigned to a nonglycosylated MRS2 isoform. The MW marker band in the left lanes is assigned a MW of 50 kDa. ( A,B ) Incubation of (0.5 M initial cell density) BRL3A cells in low (5.6 mM) or high (25 mM) glucose (Glu) for 96, 48 or 72 h. At every time point N- glycosylation, and presumably glycolysis, is enhanced at Glu (A2 vs A1, A4 vs A3, B2 vs B1). With time glycosylation is reduced, most apparently at low Glu (A3 to B1 to A1), but also at high Glu (B4 to B2). ( C ) Similarly, the reduction of Glu by altering cell number and hence Glu utilization over 72 h, reduces N- glycosylation. Four plates were seeded with 3, 1, 0.5 and 0.25 million (M) cells. The fraction of N- glycosylation increased in the same order (lanes C1, C2, C3, C4). Inhibition of glycolysis with either ( D ) galactose (Gal) or ( E ) 2-deoxyglucose (2-dG) similarly reduced the relative amount of N -glycosylation, lanes D2 vs D1 and E3 vs E2, respectively. ( F,G ) Incubation of BRL 3A cells with increasing concentrations of oligomycin (OM) (F) or rotenone (Rot) (G) decreased the relative amounts of N- glycosylation. (H) Inclusion of CoCl 2 , an inducer of a hypoxic response, resembled the results of inhibiting glycolysis. In Panels A,C,D,E and H the solid vertical lines indicate that the MW marker lane was not immediately adjacent to the lanes shown.

Article Snippet: BRL 3A rat liver fibroblasts and AFT024 mouse liver fibroblasts were purchased from the American Type Culture Collection (ATCC; Manassas, VA).

Techniques: Western Blot, Marker, Incubation, Glycoproteomics, Inhibition