Journal:

Article Title: Deacetylation of the retinoblastoma tumour suppressor protein by SIRT1

doi: 10.1042/BJ20070151

Figure Lengend Snippet: (A) Whole-cell lysates from transfected C33A cells were immunoprecipitated (IP) with the anti-Rb antibody. Complexes were resolved by SDS/PAGE, transferred on to nitrocellulose and immunoblotted using the anti-SIRT1 antibody (top panel). The blot was stripped and reprobed with the anti-Rb antibody for the presence of immunoprecipitated Rb (middle panel). Input protein representing 10% of the cell lysate is shown in the bottom panel. (B) The experiment was conducted as described in (A), except that immunoprecipitations were carried out using the anti-FLAG antibody and Western blotting with the anti-Rb antibody. (C) The experiment was conducted as described in (A and B), except that immunoprecipitations were carried out using the anti-FLAG antibody and Western blotting with either the anti-107 or anti-p130 antibody. (D) The experiment was conducted as described in (A), except that immunoprecipitations were carried out using control IgG, the anti-p107 antibody or the anti-p130 antibody and Western blotting with the anti-SIRT1 antibody. Expression of empty vector was used as a negative control for (A)–(C).

Article Snippet: Western blot analysis Collected immune complexes and cell lysates were resolved using SDS/PAGE [6, 8 or 10% (w/v) polyacrylamide gels or gradient 4–20% (w/v) Precise (Pierce) pre-cast polyacrylamide gels] and transferred on to nitrocellulose membranes (Osmonics).

Techniques: Transfection, Immunoprecipitation, SDS Page, Western Blot, Control, Expressing, Plasmid Preparation, Negative Control

Journal:

Article Title: Deacetylation of the retinoblastoma tumour suppressor protein by SIRT1

doi: 10.1042/BJ20070151

Figure Lengend Snippet: (A) Schematic representation of large-pocket Rb and mutations in the pocket region of Rb. Δ indicates mutations in the LXCXE Rb-binding site (residues 709, 713 and 757). (B) Whole-cell lysates from transfected C33A cells were immunoprecipitated (IP) with the anti-GAL4 antibody. Complexes were resolved by SDS/PAGE, transferred on to nitrocellulose and immunoblotted using the anti-SIRT1 antibody (top panel). The blot was stripped and reprobed with the anti-Rb antibody for the presence of immunoprecipitated Rb (middle panel). Input protein representing 10% of the cell lysate is shown in the bottom panel. (C) C33A cells were transfected with the indicated expression vectors. The experiment was performed as described in (B).

Article Snippet: Western blot analysis Collected immune complexes and cell lysates were resolved using SDS/PAGE [6, 8 or 10% (w/v) polyacrylamide gels or gradient 4–20% (w/v) Precise (Pierce) pre-cast polyacrylamide gels] and transferred on to nitrocellulose membranes (Osmonics).

Techniques: Binding Assay, Transfection, Immunoprecipitation, SDS Page, Expressing