3xflag peptide

HEK293T cells (4.5×106) in 10 cm diameter plates were transfected
with Halo-HuR(wt) using PEI as described above. All further steps were carried
out under red-filtered light. Following T-REX delivery of HNE as described
above, cells were lysed, debris was cleared by centrifugation
(20000×g, 10 min, 4°C), and protein levels
were normalized using Bradford assay. Halo-HuR was then
enriched by incubation with anti-Flag M2 agarose for 1 h at room temperature.
Following elution for 1.5 h with 0.3 mg/ml 3xFlag peptide
(ApexBio), eluted proteins were cleaved with TEV protease
(0.25 mg/ml final concentration) and subjected to Click coupling with Cy5-azide
as described above. The mixture was then run on 15% SDS-PAGE to resolve HuR from
Halo. In-gel fluorescence intensity signal (collected with a Bio-Rad
Chemidoc MP) was quantitated using the Gel Analysis tool of ImageJ
and T-REX targeting efficiency was calculated as previously described^{[24 (link)]} using Eqn.
3: $$\%\mathrm{Targeting}\mathrm{Efficiency}=\frac{{\text{Cy5}}_x/{\text{Coomassie}}_x}{{\text{(Cy5}}_y-{\text{Cy5}}_{\text{Halo}})/{\text{Coomassie}}_y}\times 100\%$$

SAMHD1 proteins used in nuclease assays were isolated from U937 cells transduced to express 3xFlag-SAMHD1 (WT or HD/RN) or empty vector. Cells were differentiated overnight with 10 ng/ml PMA, lysed in 50 mM Tris pH 8.0, 150 mM NaCl, 1% Triton X-100 for 20 min at 4°C and clarified at 10,000xg for 10 min at 4°C. Cleared lysates were then incubated for 2 h with anti-Flag conjugated agarose beads (EZview Red ANTI-FLAG M2 affinity gel, Sigma-Aldrich) at 4°C. The samples were then washed twice with wash buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.1% Triton X-100) and twice with assay buffer (1x PBS, 2 mM DTT, 10% glycerol, 0.01% NP-40). Bound proteins were eluted from the beads using 200 ng/μl 3xFlag peptide (ApexBio) in assay buffer. Nuclease assays were performed using a ³²P-labeled 20nt RNA probe as described in (Ryoo et al., 2014 (link)). Immunoprecipitated proteins and the probe were incubated at 37°C in assay buffer for up to 3 h. As a positive control, a sample was treated in parallel with RNaseA (40 μg/ml). Formamide loading buffer was added, the samples heated to 65°C for 5min and an aliquot of each sample was separated on a 15% denaturing urea polyacrylamide gel (SequaGel, National Diagnostics) prior to autoradiography.

pCAGIG-FLAG-SHTN1L, pCAGIG-FLAG-SHTN1S and pCAGIG-FLAG-SHTN1L^RRR>GGG constructs were individually transfected into Neuro-2a cells cultured in 15-cm dishes. Forty-eight hours after transfection, cells overexpressing the FLAG-tagged target proteins were washed with ice-cold PBS, scraped off and lysed in 5 ml buffer containing 50 mM Tris.HCl pH 7.4, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, Protease/Phosphatase inhibitor cocktails (Roche), 1 mM PMSF, and 50U/ml Benzonase (Novagen). For affinity purification, 250 µl slurry of anti-FLAG M2 magnetic beads (Sigma-Aldrich) equilibrated in lysis buffer were added into cleared cell lysate and incubated for 3 hrs at 4°C. Then collected beads on magnetic rack were washed three times with 2.5 ml lysis buffer. Bound proteins were eluted two times for 15 min at RT with 1.25 ml elution buffer containing 10 mM Tris.HCl pH 7.4, 100 mM NaCl and 200 µg/ml 3X FLAG-peptide (Apex Bio). Elutions were concentrated and washed with TBS buffer by Amicon Ultra centrifugal filters MWCO 30 kDa. Concentrated proteins and BSA standards were analyzed by polyacrylamide gel electrophoresis stained with Coomassie brilliant blue G-250 dye (Teknova) to calculate molar concentration of target molecules.

Palbociclib Isethionate (Selleckchem) was dissolved in water to 10 mM. Trametinib (Selleckchem), Rapamycin (Alfa Aesar), and SP600125 (ApexBio) were dissolved in DMSO. Human recombinant insulin was purchased from Sigma, PMA (Phorbol-12-Myristate-13-Acetate) was purchased from Acros. Anisomycin was purchased from Cayman Chemical. 3XFLAG peptide was purchased from ApexBio. All reagents were used as received.