Mouse anti actin antibody

For PARP cleavage, MTT cells were treated with the indicated dose and concentration of drug for 20 hrs at 37°C and 5% CO₂. Cleaved and full-length PARP (rabbit anti-PARP antibody from Cell Signaling and HRP-conjugated anti-rabbit antibody from Jackson ImmunoResearch) and actin (mouse anti-actin antibody from Millipore and HRP-conjugated anti-mouse antibody from Jackson ImmunoResearch), for loading control, were measured by immunoblotting.

Equal amounts of total protein confirmed by BCA-protein assay kit (Beyotime) were separated on a 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, USA). The membrane was incubated with mouse anti-Drp1 antibody (1:1000; Origene, Rockville, MD, USA), and mouse anti-actin antibody (1:500; Millipore). After washing, the membrane was incubated with horseradish peroxidase-conjugated goat anti-mouse IgG antibody (1:5000; Millipore). Immunoreactive protein bands were visualized using a chemiluminescence kit (Millipore) and quantified by Image J software.

The 293T cells were seeded in six-well plates and grown until semi-confluent. To measure the infection efficiency, cells were infected with the tested p518-L and p518-S strains in serum-free medium without trypsin) at an MOI of 2. At 0.5, 1.5, or 6 h p.i., cell lysates were collected with modified radio-immuno-precipitation assay (RIPA) buffer supplemented with dithiothreitol (DTT) (Sigma) and protease inhibitors (Millipore). Protein content was determined by the Bradford method, using Bio-Rad Protein Assay kit (Bio-Rad). Proteins in these cell lysates were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. The membranes were incubated with rabbit anti-influenza NP antibodies (GeneTex) and mouse anti-Actin antibody (Millipore), and then stained with horseradish peroxidase (HRP)-conjugated secondary antibodies specific for mouse (Jackson) and rabbit (Cell Signaling). The membranes were developed with the enhanced chemiluminescence system (Perkin Elmer), and the images were obtained by the BioSpectrum Imaging System (UVP).

Dexamethasone (Dex) was purchased from Taiji Group (Chongqing, China). Dulbecco’s Modified Eagle Medium (DMEM) was purchased from Hyclone (Logan, UT, USA). Fetal bovine serum (FBS), L-glutamine and antibiotics, mouse anti-actin antibody, goat anti-mouse IgG antibody and goat anti-rabbit IgG antibody were purchased from Millipore (Billerica, MA, USA). Mouse anti-HO-1 antibody, mouse anti-CypD antibody, rabbit anti-phospho-p38 antibody and rabbit anti-p38 antibody were purchased from abcam (Cambridge, MA, USA). Mitosox red, MitoTracker green and Lipofectamine® RNAiMAX Transfection Reagent were purchased from Thermo Scientific (Waltham, MA, USA). ON-TARGET plus Mouse ppif siRNA (L-062722) and control siRNA (D-001810) were purchased from Dharmacon Research (CO, USA). Annexin V-FITC/propidium iodide (PI) Apoptosis Detection kit was purchased from KeyGEN BioTECH (Nanjing, China). Other reagents not mentioned here were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Protein was isolated from nerve biopsies homogenized in lysis buffer (150 mM NaCl, 1 % Triton, 2 mM EDTA, 50 mM Tris, pH 7.4). 30 µg protein extract was electrophoresed on a 12 % SDS-PAGE gel and blotted for 40 min to PVDF membrane. The membranes were probed with rabbit anti-IGFBP5 (H-100, 1:5000, Santa Cruz Biotechnology) for human samples and goat anti-IGFBP5 antibody (GT15183, 1:5000, Neuromics) for murine samples in 5 % skim milk, and rabbit anti-phosho-IGF1 receptor beta (3918, 1:2000) in 5 % BSA, anti-IGF1 receptor beta (3027, 1:2000) in 5 % milk, for immunoprecipitation 1:1000 in combination with protein A agarose beads (11719386001, Roche), anti-phospho-Akt (9271, 1:2000 in 5 % BSA) and anti-Akt (9272, 1:2000 in 5 % milk, all from Cell Signaling Technology) for mouse extracts, in blocking buffer for 1 h. The appropriate HRP-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc) was used and visualized using enhanced chemiluminescence (GE Healthcare, Lifesciences). The blots were reprobed with mouse anti-actin antibody (Clone C4, 1:7000, Millipore). Film images were scanned and the intensity of IGFBP5 was standardized to mouse anti-actin. A minimum of n = 3 per group were tested in 3 independent experiments.