Anti 4e bp1 53h11

Muscle tissue (without the nervous system and motor axons or imaginal discs) was isolated from wandering third-instar larvae dissected in cold HL3. Western blot analysis was performed according to manufacturer's protocols using the following primary antibodies: rabbit anti-phospho-4E-BP (T37/46) and rabbit anti-nonphospho-4E-BP (T46), both 1:1,000 (Cell Signaling), rabbit anti-hLRRK2 (3515-1) 1:1,000 (Epitomics), rabbit anti-GFP 1:1,000 (Molecular Probes) and mouse anti-Actin 1:2,000 (Chemicon). Protein bands were visualized using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). The gel images were scanned and band intensities were quantified using MetaMorph software (Molecular Devices). The following antibodies were used for hLRRK2 overexpression and hLRRK2 short hairpin RNA (shRNA) western blottings: anti-eIF4G (2498) from Cell Signaling, mouse monoclonal anti-eIF4A was a gift from Hans Trachsel (Bern, Switzerland)66 (link), eIF4E: mouse anti-eIF4E (610270) from BD Biosciences, anti-4E-BP1 (53H11) Rabbit monoclonal antibody (9644) from Cell Signaling, anti-Phospho 4E-BP1 (236B4) Rabbit monoclonal antibody (2855) from Cell Signaling and monoclonal anti-β-actin (A5441) from Sigma.

First, cells were washed twice with PBS (Sangon Biotech), lysed in RIPA Lysis Buffer (Beyotime) supplemented with protease inhibitor cocktails and PhosStop (Roche). Protein was mixed with loading buffer, boiled and subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane (Millipore) according to a standard protocol. After blocking with 5% non-fat milk in TBST (50 mM Tris-HCl (Sigma-Aldrich) at pH 8.0, 150 mM NaCl (SinoPharm), 0.1% Tween 20 (Sigma-Aldrich)), the membrane was incubated with non-fat milk or BSA-TBST-diluted primary and secondary antibodies. Signals were detected with ECL detection reagent (Vazyme) using AI600 (GE). The antibodies used in this study were: anti-GAPDH (Proteintech, 10494-1-AP), anti-Pim-1 (D8D7Y) Rabbit mAb (Cell Signaling, 54523 S), anti-c-Myc antibody [Y69] (Abcam, ab32072), anti-c-Myc (phospho S62) antibody (Abcam, ab51156), anti-4E-BP1 (53H11) (Cell signaling, 9644 T), anti-p-4E-BP1(T32/46) (Cell signaling, 2855 T), Anti-rabit IgG, HRP-linked Antibody (Beyotime, A0208).