Glutathione sepharose 4b bead

GST fusion constructs of Pin 1 or DAPK1 were expressed in Escherichia coli and were purified using glutathione–Sepharose 4B beads (Amersham Bioscience). Equal amounts of GST or GST fusion proteins bound to glutathione–Sepharose beads were incubated with lysates from HEK 293T cells transiently transfected with BCR‐ABL‐His. The beads were washed three times, and interacting proteins were detected by Western immunoblotting.

The Rab5-binding domain (R5BD) of Rabaptin5 was first cloned into pGEX-4T-1 plasmids encoding the fusion protein GST-R5BD in Escherichia coli BL21, and expression was induced with 0.1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). GST-R5BD was then affinity purified by use of glutathione Sepharose 4B beads (Amersham Pharmacia Biotech) and quantified by SDS-PAGE. Cells were transfected with Rab5 proteins (Rab5wt, Rab5S34N, and Rab5Q79L) at 37°C for 24 h, followed by treatment with PHEV, and then collected and lysed for 15 min in lysis buffer (25 mM HEPES [pH 7.4], 100 mM NaCl, 5 mM MgCl₂, 1 mM EDTA, 1 mM dithiothreitol [DTT], 0.1% NP-40, 20% glycerol, and 0.1% protease inhibitor). To measure Rab5 GTPase activation in response to PHEV infection, the virus was preabsorbed into the cells at 37°C for 24 h, and then the cells were collected and lysed; guanosine gamma thiophosphate (GTPγS) was added to partial lysates to activate small G proteins as a positive control. After being clarified by centrifugation at 10,000 × g for 10 min at 4°C, the lysates were incubated with the purified GST-R5BD protein at 4°C overnight on a rotating mixer. The resin was subsequently rinsed with the lysis buffer and resuspended in SDS loading buffer, and Western blotting was subsequently performed.

Glutathione S-transferase (GST) recombinant proteins were expressed in E. coli strain BL21 (DE3) transformed with pGEX plasmids encoding GST or GST-PRMT5 fusion protein. Briefly, the plasmids were transformed into E. coli strain BL21 (DE3), and then cultured and induced with 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) to express GST or GST-PRMT5 fusion proteins. The GST recombinant proteins were purified with glutathione Sepharose 4B beads (Amersham Pharmacia Biotech) from the lysate of E. coli strain BL21 (DE3). Similarly, His-tagged EZH2 fusion protein was expressed in E. coli strain BL21 (DE3) and the His-tagged EZH2 fusion protein was purified with Ni-NTA agarose beads (Qiagen). GST-tagged PRMT5 or His-tagged EZH2 recombinant proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie blue to visualize His or GST fusion proteins. Subsequently, GST pulldown assays were performed to validate the direct interaction of GST-tagged PRMT5 and His-tagged EZH2 in vitro. Equal amount of GST or GST-tagged PRMT5 fusion proteins were incubated with His-tagged EZH2. After washing, the bound fusion proteins were eluted from glutathione Sepharose 4B beads, separated by SDS-PAGE and detected by immunoblotting with anti-EZH2 antibody.

pcDNA3.1 constructs with Flag tag were translated
in vitro by using the TNT-coupled transcription and translation system (Promega, Madison, USA) following the manufacturer’s instruction. GST constructs were transformed into
Escherichia coli BL21 cells and the protein expression was induced by IPTG (up to 1 mM) at 37°C for 4 h. The GST or GST fusion proteins were extracted and immobilized onto Glutathione Sepharose 4B beads (Amersham Biosciences, Piscataway, USA) at 4°C for 4 h. Equal amounts of GST and GST-fusion proteins were incubated with
in vitro translated protein at 4°C overnight. After centrifugation and three times wash with lysis buffer, the beads were eluted with 30 μL of 1×SDS loading buffer and then boiled for 10 min, followed by western blot analysis using anti-Flag-tag M2 antibody (1:5000) as the primary antibody. Ponceau stain indicated the loading of GST or GST-fusion proteins.

GST-Rab1A recombinant proteins were purified by glutathione-sepharose 4B beads (Amersham). GST and GST-Rab1A proteins were immobilized on glutathione-sepharose 4B beads and loaded with GDP or GTPγS as described earlier [32 (link),57 (link)]. CMV-ELMOD3-GFP was expressed in HEK-293T cells for 48 h. The cells were collected and lysed in buffer containing 50 mM Tris–HCl (pH8), 75 mM NaCl, 1 mM MgCl₂ and 0.05% NP-40, 100 mM sucrose, 1 mM DTT, 1xprotease Cocktail inhibitors (Roche) for 30 min at 4°C. Lysates were centrifuged, and the supernatant was incubated with GST and GDP- or GTPγS-loaded GST and GST-Rab1A for 2 h at 4°C. The samples were subsequently washed five times with lysis buffer, eluted using SDS sample buffer and analysed by SDS-PAGE.

Plasmids encoding cDNAs for the human Pals1 PDZ domain (wild-type and F318A mutant) were transformed into Escherichia coli FB810 cells and grown in LB medium at 37°C in the presence of anitibiotics. After reaching a density of A₆₀₀ = 0.6, the cells were induced with 20 µM IPTG (Sigma–Aldrich) and grown at 16°C for 18 h with agitation. The cells were harvested and resuspended in 20 mM HEPES pH 7.5 (Sigma), 100 mM NaCl (Sigma), 10 mM Benzamidine, 0.2 mM AEBSF, 1 mM DTT. The cells were disrupted by sonication and spun down at 30 000g for 30 min. Pals1^PDZ protein was extracted from the lysate using glutathione Sepharose 4B beads (Amersham Biosciences) and washed in 20 mM HEPES pH 7.5, 100 mM NaCl, 1 mM DTT, followed by removal of the GST affinity tag with GST-3C protease (PreScission Protease, Amersham Bioscience) overnight at 4°C. The eluate was further purified by size-exclusion chromatography (Superdex S75). All purification steps were performed at 4°C or on ice. Protein purity was analysed using SDS–PAGE.