E coli bl21

BIN1-SH3 cDNA generously provided by Isabelle Landrieu (University of
Lille Nord de France) was cloned into pGEX5X1 vectors using sequence and
ligation independent cloning (SLIC) (Hill and
Eaton-Rye, 2014). The BIN1-SH3 domain was amplified from the original
vector using primers 5’-TCG AGC GGC CGC ATC GTG ACA TGG GTC GTC TGG ATC
TG-3’ and 5’-AAA CGC GCG AGG CAG ATC GTC AGT TAC GGC ACA CGC TCA
GTA AAA TTC-3’, and pGEX5X1 was linearized using primers 5’-CTG
ACG ATC TGC CTC GCG-3’ and 5’-GTC ACG ATG CGG CCG CTC-3’.
SLIC products were used to transform BL21 E.Coli (New England
Biolabs, MA, USA) by heat shock. DNA was purified using QIAgen spin miniprep kit
(QIAgen, Hilden, Germany), and the cloning was confirmed by sequencing (Source
Bioscience, Nottingham, UK), using stock primers to the GST plasmid. BL21
E.coli containing either BIN1-SH3-pGEX5X1 or empty vector
pGEX5X1 were used to produce GST fusion proteins. Wild type (WT) human 2N4R tau
and PxxP mutant tau plasmids have been described previously (Lau et al., 2016 (link)). These
were expressed in HEK293 cells for 24 hours after which time cells were lysed
and the lysates used for GST pull-downs, which were performed as we described
before (Lau et al.,
2016).

For GST-SRPK2 protein purification, E. coli BL21 (New England Biolabs) was transformed with pGEX-2T-SRPK2 plasmids. The bacteria were grown at 28°C in 2x YTA medium containing 100 ug/ml ampicillin until A₆₀₀ of 0.6–0.8, followed by 100 nM IPTG treatment for 2 hr to induce protein expression. The cells were harvested by centrifugation at 6,000 g at 4°C for 15 min and re-suspended in ice-cold PBS. The cells were lysed with sonicator, followed by incubation with 1% Triton X-100 at 4°C for 30 min. Cell lysates were c leared by centrifugation at 12,000 g at 4°C for 10 min and incubated with 50% slurry of Glutathione Sepharose 4B (GE Healthcare Life Sciences) at 4°C for 2 hr. After rinsing three times with PBS, p roteins were eluted with 1 mL elution buffer (50 mM Tris-HCl and 10 mM reduced glutathione, pH 8.0).
For S6K1 in vitro kinase assay, immunoprecipitated HA-S6K1 from HEK293E was incubated with 1 µg of GST-SRPK2 in a kinase assay buffer (25 mM Tris-HCl, [pH 7.4], 10 mM MgCl₂, 5 mM β-glycerophosphate, 2 mM DTT, and 100 µM ATP) containing 5 µCi [γ-³²P]-ATP (Perkin Elmer) at 30°C for 20 min. For CK1 in vitro kinase assay, 200 ng of GST-CK1 (EMD Millipore) was incubated with GST-SRPK2 in the kinase assay buffer containing 5 µ [γ-³²P]-ATP at 30°C for 30 min. Samples were separated by SDS-PAGE, blotted onto nitrocellulose membrane, and subjected to autoradiography.