Anti puromycin antibody

Monoclonal antibodies against phospho-ERK1/2 (#8544S), ERK1/2 (#4695S), phospho-AKT (#9611S), AKT (#4685S), U0126 and cell lysis buffer were purchased from Cell Signaling Technology. Anti-Maf1 antibody (sc-365312, sc-515614) was obtained from Santa Cruz Biotechnology. DMEM/F12 and newborn bovine serum (NBS) were purchased from HyClone. Collagenase and trypsin were purchased from Gibco. Phenylephrine (PE) was purchased from Tokyo Chemical Industry. TRIzol was obtained from Sigma. RNA pol III inhibitor was purchased from Merck. Puromycin and anti-Puromycin antibody were obtained from Sigma.

SUnSET assay is modified from a previous protocol.^{46 (link)} Briefly, 4 leaf discs from 4-week-old plants were detached and floated in distilled water in 96-well plates. 50 μM puromycin was applied to samples for 40 min at each time point. Leaf discs were ground in liquid nitrogen, to which 80 μl of 2x SDS-sample buffer was subsequently added. After vigorous mixing, lysates were boiled at 95 °C for 10 min. Puromycin-labeled proteins were detected by western blot probed with an anti-puromycin antibody (Sigma).

Changes in protein synthesis after exposure to mechanical stimulation was measured by a modified method of SUnSET assay^{51 (link)}. Cells were treated with 2 µg/ml puromycin during 1 h of mechanical stimulation. Control cells were treated with 100 µg/ml cycloheximide for 5 min to stop mRNA translation prior to adding puromycin. Total protein was harvested after mechanical stretching and newly synthesized proteins were visualized by anti-puromycin antibody (Sigma-Aldrich, USA, #MABE343) by western blot.

A total of 5 × 10⁴ cells were plated onto 12-well plates, left to adhere overnight and treated with FHIN-1, AA5 and TTFA for indicated times (1, 3, or 6 hr). For the last 15 min of the treatment time, 20 μg/mL puromycin dihydrochloride (Sigma) was added to the well prior to extraction. Puromycin incorporation into neosynthesized proteins was assessd with western blotting and an anti-puromycin antibody (Sigma).

Global protein synthesis was assessed using puromycin incorporation [91 ]. An equal number of nol9^{sa1022/sa1022} and wt 120 hpf larvae were suspended in blocking solution (5% FBS / PBS), with the addition of 20 μg/ml puromycin (Sigma) and 100 μg/ml cycloheximide (Santa Cruz Biotechnology), where applicable. Larvae were dissociated by passing through 100 μm mesh and the cell suspension was incubated at room temperature for 10 min, followed by centrifugation at 2,000 rpm for 3 min. The pellets were re-suspended in blocking solution (with the addition of 100 μg/ml cycloheximide, where applicable) and incubated at 28°C for 40 min. After centrifugation, cell pellets were used for Western blot analysis using anti-puromycin antibody (1:150, kind gift from Yusuke Sekine) and anti-β-actin antibody (Sigma Aldrich, 1:1,000) for loading control. Signal strength was quantified by densitometry measurements using ImageJ. For β-actin, the single band was quantified. For puromycin, an area of gel with the weakest background (the signal in the no puromycin control) was chosen. For each lane, the full signal within the chosen area was measured and the value for the background was subtracted. The puromycin signal value was then divided by the β-actin value for the same sample.

In vivo protein synthesis rates were measured via the SUnSET technique. Two-week-old male WT and LONP1 mKO mice (4–6 mice per group) were intraperitoneal injected with 0.04 mmol/g puromycin^{61 (link)}. Thirty minute post-injection, muscles were collected and frozen in liquid Nitrogen for immunoblot analysis using anti-puromycin antibody (clone 12D10, Sigma).