affi gel blue agarose column  (Bio-Rad)

Journal:

Article Title: SKP1-SnRK protein kinase interactions mediate proteasomal binding of a plant SCF ubiquitin ligase

doi: 10.1093/emboj/20.11.2742

Figure Lengend Snippet: Fig. 3. SKP1/ASK1 and α4/PAD1 interact with C-terminal domains of AKIN10 and AKIN11. In vitro binding of SnRKs to SKP1/ASK1 is competed by PRL1, but enhanced by α4/PAD1, which selectively recruits SKP1/ASK1 and SnRK from Arabidopsis cell extracts. Unlike PRL1, SnRK is co-immunoprecipitated with SKP1/ASK1 and co-purifies with 26S proteasome. (A) Mapping of SKP1/ASK1 and α4/PAD1 binding domains of AKIN10 and AKIN11 by two-hybrid interaction assays. The results of LacZ filter assays (+ or –) indicate interactions of GBD baits, expressing different segments of AKIN10 and AKIN11 (amino acid positions are indicated in subscript), with GAD–ASK1 and GAD–PAD1 preys. Boxes indicate the positions of known SnRK domains. (B) In vitro SnRK-binding assay with SKP1/ASK1 and PRL1. A preformed [35S]AKIN10–GST–ASK1 complex was challenged with increasing amounts of MBP–PRL1. Equal aliquots from each sample were bound to GS (GST pull-down) and amylose–agarose (MBP pull-down) to monitor the amount of [35S]AKIN10 present in complex with GST–ASK1 and MBP–PRL1, respectively. Recruitment of a C-terminally truncated form of AKIN10 by MBP–PRL1 (lower band in MPB pull-down assay), but not by GST–ASK1, indicates that PRL1 can also interact with AKIN10 sequences located upstream of the C-terminal SKP1/ASK1 binding site. (C) In vitro SnRK-binding assay with SKP1/ASK1 and α4/PAD1. [35S]AKIN10 was saturated with GST–ASK1, then increasing amounts of His-α4/PAD1 were added to the samples that were bound to GS. Following SDS–PAGE separation of eluted proteins, the amounts of GST–ASK1-associated [35S]AKIN10 and His-α4/PAD1 proteins were monitored by autoradiography and western blotting with an anti-His6 antibody, respectively. (D) In vitro kinase competition assay with SKP1/ASK1 and PRL1. Upper panel, phosphorylation of TRX-KD substrate by GST–AKIN10 alone (C) and in the presence of MBP, MBP–PRL1 and GST proteins. Lower panels, GST–AKIN10 was either incubated with increasing amounts of GST–ASK1 (left panel) or pre-incubated with GST–ASK1 followed by addition of increasing amounts of MBP–PRL1 (right panel) before performing the kinase assays with the TRX-KD substrate. (E) Protein extract from Arabidopsis Col-0 cells was bound to immobilized α-ASK1 IgG and protein A–Sepharose resins. Aliquots from the cell extract and proteins eluted from the IgG matrix (IP α-ASK1) and control protein A beads (w/o IgG) were immunoblotted with α-ASK1, α-SnRK and α-PRL1 antibodies and subjected to SnRK kinase assays. (F) Protein extract from Arabidopsis Col-0 cells was bound to His-α4/PAD1 immobilized on Ni-NTA–agarose and to control Ni-NTA-resin. The cell extract (Total) and protein fractions eluted from the His-α4/PAD1 (His-α4 pull-down) and Ni-NTA (Control) beads were immunoblotted with α-SnRK and α-ASK1 antibodies. (G) Purified 26S proteasome separated and stained in a non-denaturing polyacrylamide gel (Native) was eluted for separation of subunits by SDS–PAGE, which was either silver stained (Silver) or immunoblotted with α-20S proteasome and α-SnRK antibodies. Expected molecular masses for SnRK (AKIN10 or AKIN11) and proteasomal α-subunits are indicated.

Article Snippet: Proteasomes were similarly prepared from the ASK1-HA-expressing Arabidopsis cell line, with the exception that the cleared lysate (2 g of protein) was fractionated on a DEAE-Affi-Gel blue Sepharose column (Bio-Rad) using a linear gradient of 0–500 mM NaCl in buffer E [25 mM Tris–HCl pH 7.5, 2 mM ATP, 5 mM MgCl 2 , 1 mM DTT, 10% (v/v) glycerol].

Techniques: In Vitro, Binding Assay, Immunoprecipitation, Expressing, Pull Down Assay, SDS Page, Autoradiography, Western Blot, Competitive Binding Assay, Incubation, Purification, Staining

Journal:

Article Title: SKP1-SnRK protein kinase interactions mediate proteasomal binding of a plant SCF ubiquitin ligase

doi: 10.1093/emboj/20.11.2742

Figure Lengend Snippet: Fig. 6. SnRK is associated with SKP1/ASK1, cullin and 20S proteasome α-subunits. (A) SKP1/ASK1 is specifically detected by the polyclonal α-ASK1 antibody. Double-staining of Arabidopsis cells expressing an HA epitope-tagged form of SKP1/ASK1 protein (ASK1-HA) with polyclonal α-ASK1 (red) and monoclonal anti-HA (green) antibodies shows identical images of mitotic spindles in late anaphase (left column) and phragmoplasts in telophase (right column). Chromosomes and daughter nuclei are stained with DAPI (blue). Bars = 5 µm. (B) SKP1/ASK1 is co-immunoprecipitated with SnRK and cullin. Protein extract prepared from Arabidopsis cells expressing ASK1-HA was bound to immobilized anti-HA.11 IgG. The crude extract (T) and proteins eluted from the α-HA IgG matrix (IP) were separated by SDS–PAGE and immunoblotted with α-HA, α-SnRK and α-AtCUL1 antibodies. A control immunoprecipitation experiment was performed under identical conditions with a protein extract prepared from a cell line expressing an HA epitope-tagged β-glucuronidase enzyme (HA-GUS). (C) Purification of 20S proteasome–SCF complex. 20S proteasomal fractions co-purifying with cullin, SnRK and ASK1-HA on DEAE-Affi-Gel blue and Sephacryl S-400 were immunoaffinity purified on an anti-HA.11 IgG column. Proteins eluted with HA-peptide were separated by SDS–PAGE and immunoblotted with α-20S proteasome and α-HA antibodies. (D) Immunaffinity binding to α-HA and α-SnRK IgGs destabilizes the SCF complex. A Sephacryl S-400-purified 20S proteasome–SCF fraction (TOTAL) was immunoprecipitated using immobilized α-AtCUL1, α-SnRK and α-HA IgG antibodies, separated by SDS–PAGE and immunoblotted with α-HA, α-AtCUL1 and α-SnRK antibodies.

Article Snippet: Proteasomes were similarly prepared from the ASK1-HA-expressing Arabidopsis cell line, with the exception that the cleared lysate (2 g of protein) was fractionated on a DEAE-Affi-Gel blue Sepharose column (Bio-Rad) using a linear gradient of 0–500 mM NaCl in buffer E [25 mM Tris–HCl pH 7.5, 2 mM ATP, 5 mM MgCl 2 , 1 mM DTT, 10% (v/v) glycerol].

Techniques: Double Staining, Expressing, Staining, Immunoprecipitation, SDS Page, Purification, Binding Assay