Journal: The Journal of Cell Biology
Article Title: The clathrin heavy chain isoform CHC22 functions in a novel endosomal sorting step
Figure Lengend Snippet: CHC22 is present at variable levels in most cell types and does not participate in endocytosis. (A) Detergent lysates of 10 tissue culture cell lines (equalized for protein content) were separated by SDS-PAGE and immunoblotted with antibodies against proteins indicated at the left. Tissue sources for the cell lines were cervical epithelium (HeLa), mammary gland epithelium (MDA 231), epidermal epithelium (A431), kidney epithelium (HEK 293), foreskin fibroblast (HFF), hepatocellular carcinoma (HepG2), natural killer lymphocyte (NKL), rhabdomyosarcoma (JR1), and skeletal muscle (LHCNM2 and C2C12), with all derived from human samples except C2C12 (mouse). (B) Indicated amounts of recombinant protein fragments (in ng) of CHC17 (amino acids 1–1074, CHC17-TDD) or CHC22 (amino acids 1521–1640, CHC22-TXD) and of detergent lysates (in µg) of HeLa or LHCNM2 cells (designated at the far right) were separated by SDS-PAGE and analyzed sequentially on the same immunoblot, after stripping in between, with antibodies against the CHC for the entire row indicated at the left. Below, each plot shows the intensity of the immunoblotting signal (x-axis) versus the amount of recombinant protein (left y-axis, solid squares) or detergent lysate (right y-axis, solid triangles) analyzed. CHC amount in cell lysates was determined by intersection with the immunoblotting signals from the recombinant fragments used for antibody calibration, adjusting for fragment length. Measured ratios for CHC22 compared with CHC17 were 1:8 for LHCNM2 and 1:12 for HeLa. (C) Detergent lysates of HeLa cells treated with siRNA to deplete CHCs indicated at the top or with control siRNA were separated by SDS-PAGE and immunoblotted with antibodies against proteins indicated at the left. α-Tubulin (α-tub) serves as a loading control. (D–F) HeLa cells treated with control siRNA (D) or siRNA to deplete cells of CHC17 (E) and CHC22 (F) were incubated with fluorescent transferrin (green in merged insets) and internalization was allowed for 10 min. Cells were then fixed and processed for double immunofluorescence using antibodies against CHC17 (red in merged insets) or CHC22 (blue in merged insets) as indicated. Bars, 20 µm.
Article Snippet: LHCNM2 human skeletal muscle cells ( ) were maintained in tissue culture flasks coated with rat-tail collagen in basal medium (4:1 DME/M199, 0.02 M Hepes, 0.03 µg/ml ZnSO4 , and 1.4 µg/ml vitamin B12) with 15% fetal bovine serum (FBS), 55 ng/ml dexamethasone, 2.5 ng/ml hepatocyte growth factor (Sigma-Aldrich), 50 U/ml penicillin, 50 µg/ml streptomycin, and 0.625 µg/ml fungizone (growth medium).
Techniques: SDS Page, Multiple Displacement Amplification, Derivative Assay, Recombinant, Stripping Membranes, Incubation, Immunofluorescence