Journal: Oncogene

Article Title: HER2 drives Mucin-like 1 to control proliferation in breast cancer cells.

doi: 10.1038/onc.2015.487

Figure Lengend Snippet: Figure 3. MUCL1 RNA and protein expression was examined in a panel of breast and lung cancer cell lines as described in Table 1. (a) KPL4 cells were transiently transfected with either NT siRNA or MUCL1 siRNA for 48 h. Cell lysates were probed using a rabbit polyclonal anti-MUCL1 antibody. β-Actin was used as a loading control. (b) HEK-293 cells were transfected with a DDK-tagged MUCL1 expression vector or empty vector. After 48 h, cell lysates and culture supernatant were immunoblotted using anti-DDK and anti-MUCL1 antibodies. (c) MDA-MB-361 cells were transiently transfected with either NT siRNA or MUCL1 siRNA for 48 h. Cell media was changed and conditioned media was collected 48 h later and assessed by MUCL1 enzyme-linked immunosorbent assay (ELISA). *Po0.01 (n = 3). Cell lysates were probed using an anti- MUCL1 antibody. (d) RNA was extracted from cells and assessed for MUCL1 expression by reverse transcription PCR. RNA expression is shown as fold expression in each cell line divided by the median of MUCL1 expression across the panel of cell lines ± s.d. (n = 3 technical replicates). For assessing protein levels, cells were grown for 48 h in serum-free media. Culture supernatant was collected and the secreted MUCL1 was measured by ELISA and normalized to the cell number. Intracellular MUCL1 levels were examined in whole-cell lysates by western blotting for comparison. The experiments were repeated twice with similar results.

Article Snippet: MUCL1 TruORF cDNA clone in pLenti-C-Myc-DDK, pCMV6-Entry vector and DDK antibody were from Origene (Rockville, MD, USA).

Techniques: Expressing, Transfection, Control, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Reverse Transcription, RNA Expression, Western Blot, Comparison

Journal: Oncogene

Article Title: HER2 drives Mucin-like 1 to control proliferation in breast cancer cells.

doi: 10.1038/onc.2015.487

Figure Lengend Snippet: Figure 4. (a) HEK-293 cells were transfected with a DDK-tagged MUCL1 expression vector or empty vector. After 48 h, live cells were stained with anti-DDK or anti-MUCL1 antibodies followed by staining with a fluorescently labeled secondary antibody and analyzed by FCM. Cells expressing a known DDK-tagged membrane protein (positive control) demonstrated a significant shift in fluorescence. No shift in fluorescence was detected in HEK-293 cells transfected with DDK-tagged MUCL1 by either antibody. (b) HEK-293 cells were transfected with a DDK-tagged MUCL1 expression vector or empty vector. After 48 h, DDK-tagged MUCL1 was probed for using the ThermoScientific Cell Surface Protein Isolation Kit. Cells were treated with a biotinylation reagent to label surface proteins and then collected, lysed and labeled proteins were purified using neutravidin agarose resin. The eluate contains the isolated, labeled cell surface proteins and the flow-through (FT) contains unlabeled, intracellular proteins. The experiments were repeated twice with similar results.

Article Snippet: MUCL1 TruORF cDNA clone in pLenti-C-Myc-DDK, pCMV6-Entry vector and DDK antibody were from Origene (Rockville, MD, USA).

Techniques: Transfection, Expressing, Plasmid Preparation, Staining, Labeling, Membrane, Positive Control, Isolation