Complete protease inhibitor tablet

To investigate interactions between HuR and target mRNAs, RNA immunoprecipitation (RIP) was performed on C2C12 myotube lysate according to standard methods (43 (link),44 (link)). Briefly, C2C12 myotubes grown in a 150 cm dish were rinsed twice with ice-cold PBS and lysed with an equal pellet volume of RIPA-2 buffer [50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% Na-deoxycholate, 1 mM EDTA and 1 cOmplete protease inhibitor tablet (Sigma)]. Protein-A Dynabeads (Invitrogen) were incubated with either mouse IgG or HuR antibody (27 (link)). Beads coated in antibody were resuspended in NT2 buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl₂, 0.05% Nonidet P-40 with Rnasin [Promega] and 1 mM DTT). Thawed and clarified lysates were added and the bead/antibody/lysate mixture was incubated at 4°C overnight rotating end-over-end. Beads were washed with cold NT2 buffer five times. Proteinase K treatment released RNAs from bound proteins and input and bound RNA was isolated with TRIzol (Invitrogen) and reverse transcribed as described above. Enrichment for Pabpn1 and Gapdh transcripts with HuR immunoprecipitation was calculated using the comparative Ct method (29 (link)), with samples normalized to input and compared to IgG control. Data are presented as fold enrichment relative to Gapdh enrichment for each sample.

UV-crosslinking and immunoprecipitation of RNA was performed using standard methods (46 (link)). Briefly, myoblasts grown in 100 mm collagen coated dishes, rinsed twice with ice-cold PBS, crosslinked with UV (254 nm) and lysed with an equal pellet volume of RIPA-2 buffer [50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% Na-deoxycholate, 1 mM EDTA, RNase OUT (Invitrogen), and 1 cOmplete protease inhibitor tablet (Sigma)]. Protein A-Dynabeads (Invitrogen) were incubated with either rabbit IgG or MATR3 antibody (Bethyl Labs). Beads coated in antibody were resuspended in NT2 buffer [50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl₂, 0.05% Nonidet P-40] supplemented with RNase OUT (Invitrogen) and 1 mM DTT. Thawed and clarified cell lysates were added, and the bead/antibody/cell lysate mixture was incubated at 4°C overnight while tumbling end-over-end. After incubation, beads were magnetized and washed five times with cold NT2 buffer. RNA was released from bound proteins by Proteinase K treatment and input and bound RNA was isolated with TRIzol (Invitrogen) according to the manufacturer's instructions.

Naproxen (>99% purity; TCI America, Oregon, USA), EDTA, EGTA, Sodium orthovanadate, Igepal CA-630, Triton X-100,) Complete Protease inhibitor tablet and PhosSTOP phosphate inhibit tablet (Sigma) were purchased. Primary antibodies against PCNA (ab-29), cyclin D1 (ab-134175), caspase-9 (ab-52298), and IL-10 (ab-133575) were purchased from Abcam (MA, USA). Antibodies against caspase-3 (cs-9662), p21 (cs-2947), PARP (cs-9542), actin (cs-4970), cre (cs-15036s), RalA (cs-4799s), PI3K (cs-4292s), and horseradish-peroxidase-conjugated secondary antibodies (anti-rabbit, -goat, and –mouse) were purchased from Cell Signaling Technology (Beverly, MA, USA). Primary antibody against p21 (sc-397), caspase-3 (sc-397; for immunohistochemistry [IHC]), and COX-2 (sc-7951) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Prostaglandin E₂ ELISA kit (Cat. No. 514010) and mouse CXC-Chemokine receptor 4 (CXCR4) ELISA kit (Cat. No. MBS701736) were purchased from Cayman Chemical Company (Ann Arbor, MI) and MyBiosource.com (San Diego, USA). The Histostain-Plus 3^rd Gen IHC Detection Kit (Life Technologies, NY, USA) and DeadEnd Colorimetric TUNEL system (Promega, Wisconsin, USA) were purchased.