Table 1.
Antibodies used for flow cytometry and immunoblotting.
Table 2.
Oligonucleotide sequences of the GPBB shRNAs.
Fig 1.
Expression of Ligab antigen in liver progenitor cells.
Two liver progenitor cells, Lig-8 (A) and WB-F344 (B) and two liver non-progenitor cells, MF (C) and H4IIE (D) were examined for Ligab antigen expression with flow cytometry. The cells were incubated with the Ligab antibody (red line) or control mouse IgG (grey dotted line) followed by FITC-conjugated secondary antibody staining before flow cytometry analysis. Numbers indicated percent of cells with fluorescence intensity greater than 101.
Fig 2.
Silver-staining of the Ligab immunoprecipitates of Lig-8 cells.
Membrane and cytosolic fractions of the Lig-8 cells were precipitated with the Ligab antibody and subjected to SDS-PAGE separation followed by silver staining. Mouse IgG immunoprecipitates served as a negative control. A unique band at about 38 kDa in the cytosolic and membrane fractions (arrows) were excised and mixed for protein identification by LC-MS/MS. Extract fr.: Extract of protein fraction; NP: no protein.
Fig 3.
Expression of GP isoforms in liver progenitor cells.
Cell lysates from two liver progenitor cells WB-F344 and Lig-8 (A) and two non-progenitor cells MF and H4IIE (B) were analyzed with immunoblotting for the expression of GPBB (brain isoform glycogen phosphorylase), GPLL (liver isoform glycogen phosphorylase) and GPMM (muscle isoform glycogen phosphorylase). Protein extracts from rat brain, liver and muscle served as positive controls for the GPBB, GPLL and GPMM antibodies.
Fig 4.
Ligab immunoreactivities in WB-F344 and Lig-8 cells with or without shRNA-mediated GPBB knockdown.
(A) Control (shControl) or lentiviruses expressing shRNA (shRNA3119 or shRNA3759) targeting GPBB was used to knockdown GPBB in WB-F344 and Lig-8 cells. After puromycin selection, control and stable GPBB knockdown cells were analyzed for GPBB expression with immunoblotting. The top panel shows the representative immunoblotting results. The bottom panel summarizes the results from 3 experiments. Y-axis: Relative quantity, intensities of protein band over that of shControl. (B) Percentages of Ligab reactive cells and mean fluorescence intensity (MI) by Ligab staining in Lig-8 and WB-F344 cells. Values are mean ± SEM.
Fig 5.
Detection of GPBB in the Ligab immunoprecipitates.
Ligab immunoprecipitates were prepared from indicated cell extracts, separated with SDS-PAGE and immunoblotted with the GPBB antibody.
Fig 6.
Time course expression of GPBB, GPLL and CK19 in WB-F344 cells during sodium butyrate (SB)-induced differentiation.
(A) shows representative immunoblotting results and (B) shows a summary of 3 independent immunoblotting results. The cells were plated onto dishes on day 0 and SB was added into culture conditions on day 1. Y-axis: quantity, intensity of interest protein band over that of β-actin. Values are mean ± SEM. * P < 0.05, Student’s test against values of the day 1.
Fig 7.
Expression of CK19 in GPBB knockdown WB-F344 cells with sodium butyrate (SB)-induced differentiation.
The cells were cultured in the presence of 5 mM sodium butyrate for 2 or 3 days before analyzed for mature cell marker CK19 expression with indirect immunofluorescence staining followed by flow cytometry. Numbers indicate percent of the cells with fluorescence intensity greater than 101. Red line, cells stained with the CK19 antibody; grey dotted line, cells stained with the control mouse IgG.
Fig 8.
Survival of GPBB knockdown WB-F344 and Lig-8 cells under low glucose conditions.
Control or lentivirus expressing shRNA3119 or shRNA3759 targeting GPBB was used to infect Lig-8 (A) and WB-F344 (B) cells. After puromycin selection, stably knockdown cells were cultured in mediums with various levels of glucose: high (4.5 g/L), low (1.0 g/L) or zero glucose/pyruvate for 24 hours. All cells in the supernatant and those attached to the plate were harvested and mixed. The nuclei were stained with propidium iodide before flow cytometry analysis. Cells with nucleus staining intensity less than those of the G0/G1 phase cells were considered apoptotic. Values are mean ± SEM. * P < 0.05 and ** P < 0.01, Student’s test against values of the control knockdown (shControl).