Journal: Vision (Basel, Switzerland)

Article Title: Dexamethasone Modulates the Dynamics of Wnt Signaling in Human Trabecular Meshwork Cells.

doi: 10.3390/vision7020043

Figure Lengend Snippet: Figure 2. Myocilin protein expression of DEX-treated HTM cells over 10-day time course. Western blot analysis showed the 10-day study of DEX-induced myocilin protein levels for cell batches HTM6 and HTM13. HTM cells were treated with DEX (100 nM) for 10 days. Each day, two flasks were randomly selected and harvested to isolate cytoplasmic fractions. These cytoplasmic fractions were analyzed by Western blot with antibodies against myocilin and β-actin. B-actin was used as a control and for normalization. Images of the gels are presented in (A,B). The background contrast was adjusted, and blots were quantified for fold change (f.c.) using the ImageJ software. Western blot analysis showed that myocilin protein levels began increasing on day 2, and a similar observation was made for both batches. Myocilin protein levels first peaked on day 6 for HTM6 and for HTM13 (C,D). Myocilin protein levels dropped on day 9 for both DEX-treated HTM cell batches (C,D). Overlay of quantitative qPCR and Western blot analysis, as shown by normalized change (n.c.), indicates that there was a correlation between AXIN2 and myocilin expression. Overlay of DEX-induced MYOC and AXIN2 gene expression and myocilin protein expression of HTM6 and HTM13 over 10-day time course (E,F). Over 10 days, AXIN2 and MYOC expression was enhanced; however, there was an increase in MYOC expression on day 8 and day 9 and a sharp decline of AXIN2 gene expression and myocilin protein expression on day 9.

Article Snippet: Then, at room temperature, the loaded membranes were blocked with Superblock (Bio-Rad Laboratories) and incubated for approximately 60 min. After incubation at 4 ◦C, the loaded membranes were incubated with primary anti-myocilin antibody (1:500) and anti-β-actin (1:2000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight.

Techniques: Expressing, Western Blot, Control, Software, Gene Expression

Journal: PLoS ONE

Article Title: Cellular Processing of Myocilin

doi: 10.1371/journal.pone.0092845

Figure Lengend Snippet: Total lysates from normal human TM cells without or with the LCT treatment were immunoprecipitated (IP) with rabbit anti-myocilin polyclonal antibody or normal rabbit IgG (as a negative control, NC) followed by immunoblotting (IB) with mouse anti-ubiquitin monoclonal antibody. Myocilin pull down by rabbit anti-myocilin (MYOC), but not the rabbit IgG control, showed multiple bands immunoreactive to anti-ubiquitin (left panel). The intensity of the ubiquitin-positive bands was enhanced by prior LCT treatment. The same blot was also probed with anti-myocilin (right panel) to verify the IP procedure. Arrow head, the 55/57-kDa myocilin bands.

Article Snippet: Lysates from human TM cells untreated or treated with 1 μM LCT for 16 h were immunoblotted using polyclonal anti-myocilin or monoclonal anti-ubiquitin (1∶2000, Biomol, Enzo Life Sciences).

Techniques: Immunoprecipitation, Negative Control, Western Blot, Ubiquitin Proteomics, Control