Journal: Cell reports

Article Title: Non-acylated Wnts can promote signaling

doi: 10.1016/j.celrep.2018.12.104

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Each IP sample was pre-cleared at 4˚C for 1 h with 10 μl settled protein-G agarose (ThermoFisher Scientific) and incubated, rotating, overnight at 4˚C with 1 μg 9E10 α-myc antibody (UPenn Cell Center).

Techniques: Recombinant, Protease Inhibitor, Luciferase, SYBR Green Assay, Affinity Purification, Expressing, Software

Journal:

Article Title: Association of a protein phosphatase 1 activity with the human factor C1 (HCF) complex

doi:

Figure Lengend Snippet: Recombinant HCF binds PP1. The HCF cDNA fragment in clone 17 was expressed as a β-galactosidase (β-Gal) and GST fusions. (A) Western blot of an SDS–PAGE gel probed with digoxigenin-labelled PP1-γ. The arrowhead indicates recombinant fusion protein. The small arrow indicates a proteolytic product of β-Gal–HCF. Escherichia coli cells were grown to an OD600 of 0.9–1. Each lane contained 10 µl of a 25 times concentrated E.coli lysate. Lane 1 contains bacterial lysate of cells expressing β-Gal alone. Lanes 2 and 3 contain bacterial lysates of cells expressing HCF (clone 17) and NIPP1 fusions respectively. (B) The GST–HCF fusion protein was incubated with purified PP1 (~3 nmol of each protein) and glutathione–Sepharose beads. Each GST-fusion pull-down was done in duplicate. After several washes the samples were resolved by SDS–PAGE and immunoblotted with anti-PP1. Lanes 1 and 2 contained the binding assay between the GST–HCF fragment in clone 17 and PP1-γ. Lanes 3 and 4 contained GST and PP1 and lanes 5 and 6 contained a positive control: GST-p53BP2 (p53BP2 is known to bind PP1 avidly) (33). The arrow indicates bands corresponding to the PP1 pulled down. (C) HCF was immunoprecipitated from 0.4–0.5 mg of HeLa nuclear extract using ~5 µg of anti-HCF antibody and the beads resuspended in ~50 µl of SDS–PAGE loading buffer. Fifteen microlitres of the immunoprecipitates of HCF from HeLa nuclear extract were electroblotted as above and probed with anti-HCF antibody (upper panel) and anti-PP1 antibody (lower panel). Each immunoprecipitation experiment contained duplicate samples. Lanes 1 and 2 contained nuclear extract. Lanes 3 and 4 had immunoprecipitates using pre-immune IgG (PI) and lanes 5 and 6 represent immunoprecipitates using anti-HCF antibody. The brace indicates the major HCF polypeptides immunoprecipitated by anti-HCF antibody. The arrow indicates the PP1 co-immunoprecipitated with the HCF polypeptides.

Article Snippet: 100 µl of nuclear extract (4–5 mg/ml) was precleared for 1 h at 4°C on 25 µl of settled protein G Sepharose (Amersham-Pharmacia) or protein G agarose beads (Roche) that had been preincubated with ~10 µg of sheep pre-immune IgG.

Techniques: Recombinant, Western Blot, SDS Page, Expressing, Incubation, Purification, Binding Assay, Positive Control, Immunoprecipitation