rabbit polyclonal anti-vigilin  (Santa Cruz Biotechnology)

Journal:

Article Title: RNAi-mediated depletion of the 15 KH domain protein, vigilin, induces death of dividing and non-dividing human cells but does not initially inhibit protein synthesis

doi: 10.1093/nar/gkg768

Figure Lengend Snippet: Rapid disappearance of vigilin in cells transfected with vigilin-specific siRNA. (A) HeLa cells were either not transfected (Untrans), mock transfected (Mock), transfected with either of two vigilin-specific siRNAs (Vig 1 and Vig 2), or with a non-specific pGL3 luciferase-specific siRNA (pGL3). Forty-eight hours after transfection, the cells were harvested, extracts were prepared, and 10 µg of each sample was fractionated on an 8% polyacrylamide gel and analyzed for vigilin content by western blotting with polyclonal rabbit anti-vigilin antibody. Purified recombinant FLAG epitope-tagged vigilin (fVig) was run as a control. (B) HeLa cells were left untransfected (Untrans), mock transfected (Mock), or were transfected with Vig 2 siRNA (0.84 µg) or with increasing concentrations of control pGL3 siRNA (0.14–0.84 µg). Forty-eight hours post-transfection, cell extracts were prepared, and 10 µg of each sample was fractionated by SDS–PAGE and analyzed for vigilin by western blotting. (C) HeLa cells were transfected with pGL3 or Vig 2 siRNAs, cells were harvested at 12, 24, 36 and 48 h post-transfection, extracts were prepared, and 30 µg of each sample was fractionated on an 8% polyacrylamide gel and analyzed for vigilin by western blotting. As a control for the specificity of the siRNAs, 20 µg of each extract was run on a 12% SDS–PAGE gel and analyzed for actin content by western blotting.

Article Snippet: The membranes were blocked overnight at 4°C in 1% non-fat dry milk (1 h at room temperature in 5% non-fat dry milk for actin and PARP western blots), then probed with either rabbit polyclonal anti-vigilin, or anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or anti-PARP (Cell Signaling, Beverly, MA, USA) antibody for 1 h at room temperature (overnight at 4°C for actin and PARP), washed and probed with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature.

Techniques: Transfection, Luciferase, Western Blot, Purification, Recombinant, FLAG-tag, Control, SDS Page

Journal:

Article Title: RNAi-mediated depletion of the 15 KH domain protein, vigilin, induces death of dividing and non-dividing human cells but does not initially inhibit protein synthesis

doi: 10.1093/nar/gkg768

Figure Lengend Snippet: Vigilin knockdown in HeLa cells results in programmed cell death. HeLa cells were treated with 6.25 nM etoposide (a standard inducer of programmed cell death), mock transfected, or transfected with pGL3 or Vig 2 siRNAs. A western blot was performed 48 h post-transfection using a polyclonal antibody to poly(ADP-ribose) polymerase (PARP). A western blot using a polyclonal antibody to actin was also performed as a loading standard. Because the etoposide-treated cells were in the late stages of apoptosis, they exhibited reduced levels of actin. Levels of actin in the pGL3 and Vig 2-treated cells were similar and the ratio of the cleaved PARP to uncleaved PARP is dramatically increased in the Vig 2-treated cells relative to the mock or pGL3-treated cells.

Article Snippet: The membranes were blocked overnight at 4°C in 1% non-fat dry milk (1 h at room temperature in 5% non-fat dry milk for actin and PARP western blots), then probed with either rabbit polyclonal anti-vigilin, or anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or anti-PARP (Cell Signaling, Beverly, MA, USA) antibody for 1 h at room temperature (overnight at 4°C for actin and PARP), washed and probed with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature.

Techniques: Knockdown, Transfection, Western Blot