α flag m2 antibody

Stably transfected Flp-In™ T-REx™ 293 cell lines (Thermo Fisher Scientific R78007) for the tetracycline-inducible expression of TRIP4 variants with N-terminal 2xFlag-His₆ or C-terminal His₆-2xFlag tags were generated according to the manufacturer’s guidelines^{10 (link)}. Transfection of the parental cell line was done using X-tremeGENE HP DNA Transfection Reagent (Sigma-Aldrich). After hygromycin-based selection of cells that had genomically integrated the expression cassette, tetracycline-induced expression of the tagged proteins was confirmed by western blotting using a monoclonal α-Flag-M2 antibody (Sigma-Aldrich F3165; 1:7500). For expression of HA-tagged TRIP4 variants, the pHAGE-HA-trip4 vectors encoding HA-tagged TRIP4^wt, TRIP4^ΔZnF, TRIP4^{L174A-L180A-I190A} or TRIP4^C171A-C184A, were transfected into 293T cells (ATCC CRL-3216) using Transit293 transfection reagent (Mirus Bio).

The sub-cellular localizations of the Flag/His-tagged versions of TRIP4 were determined by immunofluorescence^{51 (link)}. 293T cells expressing Flag-tagged TRIP4 variants were grown on coverslips and fixed using 4% (v/v) paraformaldehyde for 20 min before permeabilization using 0.1% (v/v) Triton-X-100 in PBS for 20 min. Cells were blocked using PBS supplemented with 10% (v/v) fetal bovine serum (FBS) and 0.1% (v/v) Triton-X-100 for 1 h, then treated for 2 h with an FITC-conjugated α-Flag-M2 antibody (Sigma-Aldrich F4049; 1:200) diluted in PBS containing 10% FBS and 0.1% Triton-X-100. Cells were washed, and coverslips were mounted using mounting media containing DAPI. Cells were imaged using a Nikon Ti2 2-E inverted microscope.

Protocol used for CLIP assay from pFlareG plasmid was essentially as in the previous section with some modifications. Briefly, HEK293T cells were transfected with pFlareG reporter in combination with plasmid vectors expressing FLAG-PTBP2 proteins and HA-KIS proteins. After crosslinking, cells were resuspended in lysis buffer plus 40 U/ml RNase inhibitor (Attendbio, RNI-G), sonicated and incubated with α-FLAG M2 affinity gel beads overnight at 4°C. Beads were washed twice with lysis buffer, twice with lysis buffer without detergents and once with DNase buffer (10 mM Tris–HCl, pH 7.4, 2.5 mM MgCl₂, 0.5 mM CaCl₂). Beads were digested with DNase I (Merck-Sigma, DN25) for 15 min and inactivated. RNAs bound to the beads were used as a template for cDNA synthesis using Maxima H Minus cDNA first strand synthesis Kit (Thermo Fisher, 10338179) and random hexamers as primers. 2 µl of cDNA was used as a template in a real-time qPCR assay. pFlareG mRNA was amplified with GFP annealing primers (see Supplementary file 3). Relative levels of pFlareG were calculated for each condition using GAPDH as a housekeeping gene and normalized relative to FLAG-PTBP2 protein levels immunoprecipitated checked by western blot using α-FLAG M2 antibody (1:500; Merck-Sigma, F3165) and IRDye 800RD (1:10,000; LI-COR Biosciences, 926-32210).

For co-immunoprecipitation experiments, HEK-293T cells were harvested 48 h after transfection. The cells were re-suspended in RIPA buffer (50 mM Tris pH7.4, 150 mM NaCl, 1% NP-40, 0.1% sodium deoxycholate, 0.1% SDS, 5 mM EDTA, protease and phosphatase inhibitors), rocked 1 h at 4 °C, and spun for 5 min at 600 g. The supernatants were incubated with protein G magnetic beads (Dynabeads, Invitrogen) for 30′ at 4 °C. After the preclearing, the samples were incubated with 2 μg of αFlag M2 antibody (Sigma) overnight and precipitated with protein G dynabeads. IP of anti-phospho-Threonine–Proline antibody (#9391, Cell Signalling) was performed similarly, but using protein A dynabeads. The beads were collected and washed three times with 1 mL of cold RIPA buffer, and bound proteins were separated by SDS-PAGE gels and visualised by western blots.
IP of endogenous α4 was carried out in hippocampus of adult female mice (≈ 60 days old) with a polyclonal antibody (12979-1-AP, Proteintech). Briefly, cleared cell extracts in RIPA buffer (with protease and phosphatase inhibitors) were immuno-precipitated with Protein A linked to magnetic beads (Invitrogen). An anti-Flag (#F7425, Sigma) was used as a mock control. Washes were performed with RIPA buffer without SDS.

HEK293T cells were transfected with a plasmid encoding full-length GpA protein with a Flag M2 tag in the amino terminus (after GpA signal peptide and cleavage site of the protease). Cells were also co-transfected with a plasmid encoding alternatively the ER or PM markers. Forty-eight hours after transfection cells were fixed (4% formaldehyde in PBS, 15 min) and washed in PBS (x3). Permeabilization was done with PBS, 1% BSA, 0.1% Triton X-100 for 2 minutes. Immuno-stainings were done using a primary α-Flag M2 antibody (Sigma), followed by a secondary Alexa647-conjugated anti-Mouse antibody (Sigma). Additionally, cells were DAPI stained for nucleus staining.