Monoclonal flag antibody

Following titration experiment results, all experiments in slices were performed at a dilution of approximately 1 QD to 10 anti-GFP. QD-antibody complexes were prepared by incubating 3 μl of QD655 Goat F(ab')2 anti-Rabbit IgG (Molecular Probes, #Q-11421MP) with either 3 μl of GFP Rabbit Serum Polyclonal Antibody (Molecular Probes, #A6455) or 3 μl of FLAG Monoclonal Antibody (Sigma-Aldrich, #2555). The cocktail was then diluted in 54 μl of PBS (final QD concentration of 50 nM), vortexed, briefly spun down and incubated for 30 min at room temperature.

We followed essentially the procedures established by Cochrane et al.^{22 (link)} with minor modifications. Briefly, NMVMs were lysed in RIPA lysis buffer (50 mM Tris/pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride, EDTA, leupeptin, and protease inhibitors) supplemented with protease inhibitors and the lysate was immunoprecipitated with a monoclonal Flag antibody (Sigma, USA) at 4 °C. As a negative control, an equal amount of the lysate was immunoprecipitated with a monoclonal GST antibody (Santa Cruz Biotechnology, CA, USA). The immune complexes were collected with protein A/G plus-agarose beads (Santa Cruz Biotechnology, CA) and washed ten times with PBS. Flag-CIP85 and Cx43 were detected by immunoblotting. The physical interaction between endogenous CIP85 and Cx43 was explored by precipitating with the CIP85 antibody or a polyclonal Flag antibody (Santa Cruz Biotechnology, CA, USA) as a negative control.

Y. pestis wild-type strain 201 belongs to a newly established Y. pestis biovar, microtus, which is avirulent in humans but highly lethal in mice. The hfq deletion strain (Δhfq) was generated by λ-Red homologous recombination methods as previously described (38 (link)). The Y. pestis Hfq-FLAG, Hfq, and WT-FLAG strains were constructed by transforming the pHfq-FLAG and pHfq into the Δhfq strain and Flag into the WT strain, respectively. Bacteria were grown in brain heart infusion (BHI) broth (Difco) supplemented with appropriate antibiotics overnight at 26°C with shaking at 200 rpm until exponential growth phase (optical density at 629 nm [OD₆₂₀] of 0.8). Bacterial growth was stopped by centrifugation for 6 min at 5,000 rpm at 4°C. The pellets were frozen into liquid nitrogen and stored at −80°C until the cells were lysed. Western blotting was performed by using monoclonal FLAG antibody (Sigma) to detect the FLAG-tagged proteins.

Briefly, to prepare the total cell lysates, normal and ZFP36-overexpressing HeLa cells were lysed in RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1.0% deoxycholate, 1% Triton X-100, 1 mM EDTA, and 0.1% SDS. The samples were centrifuged (12,000×g for 5 min) and the supernatants were further analyzed on a 10% SDS-PAGE gel and subsequently transferred onto a PVDF membrane (Millipore). TTP was detected using a monoclonal Flag antibody (Sigma-Aldrich) diluted in TBST (1:2000) and Action (Abclonal) was used as the loading control (1:2000).

Proteins from normal and EIF5A2-OE SH-SY5Y cells were separated by electrophoresis on 10% sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel and subsequently transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, San Jose, CA, USA). The blots were cut prior to hybridisation with antibodies. The overexpression of EIF5A2 was detected using a monoclonal Flag antibody (Sigma-Aldrich) diluted in tris-buffered saline tween (TBST) (1:2000) and GAPDH was used as the loading control.

Monoclonal Arc antibody (E-7 & C-7), monoclonal HSP27 antibody, monoclonal α tubulin antibody, monoclonal β-actin antibody and monoclonal GST antibody were purchased from Santa Cruz: polyclonal GAPDH antibody, monoclonal HA antibody and polyclonal Prx6 antibody from Ab Frontier, Inc.: monoclonal Hsc/Hsp70 antibody, monoclonal HSF1 antibody and monoclonal PARP-1 antibody from Enzo: monoclonal FLAG antibody from Sigma Aldrich: monoclonal Lamin B antibody from Calbiochem. Hydrogen peroxide was purchased from Samchun Pure Chemical Co. and diamide, sodium arsenite and cycloheximide from Sigma Aldrich.