Immobilon p pvdf

Mice were anesthetized with isoflurane and perfused with saline before tissues were collected and flash frozen in liquid nitrogen. Tissues were homogenized and sonicated in RIPA buffer containing HALT protease-phosphatase inhibitor (Thermo Fisher 78430) and 0.625 mg /mL N-ethylmaleimide (Sigma E3876). Protein concentrations were determined by DC-protein assay (BioRad) and normalized. Proteins were separated on NuPAGE 4–12% Bis-Tris Protein Gels (Thermo Fisher NP0336BOX) and transferred to Immobilon-P PVDF (0.45-μm pore size, Merck Millipore). Immunoreactivity was detected with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher) and an iBright (Thermo Fisher). Images were quantified on ImageJ, with band intensity being normalized to the indicated loading control.

Cells grown in M9 minimal media (0.5 M sorbitol) and normalized by OD_600nm, lysed in Laemmli SDS-loading dye and separated in 15% acrylamide gels and transferred to 0.45 μm hydrophobic Immobilon-P PVDF membrane (Merck Millipore). Immunoblotting was as described previously (Baba et al., 2006 (link)). Rabbit primary antibodies; α-Lpp antibody (kindly provided by T. Silhavy) and α-OmpA were diluted 1:400,000 and 1: 30,000, respectively in 5% skim milk, TBST. The membranes were incubated with goat, α-rabbit IgG, HRP-conjugated secondary antibody (Sigma; 1: 20,000 in 5% skim milk, TBST), and washed with TBST. Detection was by enhanced chemiluminescence with ECL prime western blotting detection reagent (GE Healthcare Life Sciences), visualized using Super RX-N film (Fujifilm).

Equal amounts of total protein were run on 8% polyacrylamide gels at 70–90 volts. Proteins were transferred to Immobilon-P PVDF membranes (Merck Millipore, Burlington, MA, USA) for 2 h at 1000 mA and blocked at RT for 1 h in 5% non-fat milk diluted in TBS 1× (25 mM Tris-HCl, 150 mM NaCl) with 0.1% Tween-20 (TBS-T), following primary antibody anti-PGRN (1:1000, Abcam ab208777, Cambridge, UK) incubation at 4 °C overnight. After 3 washes in TBS-T, the membranes were incubated with the appropriate secondary antibody (1:10,000) at RT for 2 h. The membranes were then washed 3 times in TBS-T and incubated with Vistra EFC (Enhanced Chemifluorescence) substrate (Merk, Darmstadt, Germany ) for 5 min at RT. The fluorescence signal was visualized using a ChemiDoc System (Bio-Rad, Hercules, CA, USA) and analysis was performed using the Image Lab Software (Bio-Rad, Hercules, CA, USA). Anti-GAPDH (1:5000, Merck Millipore MAB374, Burlington, MA, USA) was used as a loading control. The secondary antibodies employed were alkaline phosphatase affinipure goat anti-mouse IgG (Jackson Immunoresearch, #115-055-146, West Grove, PA, USA) and alkaline phosphatase affinipure mouse anti-rabbit IgG (Jackson Immunoresearch, #211-055-109, West Grove, PA, USA).

Western blot analysis was performed as described previously [28 (link)]. Briefly, U251 MG cells were treated with test compounds or inhibitor (KC7F2, Selleckchem, Houston, TX, USA). Western blot samples were obtained by scraping 10⁶ cells from 6-well plates in 250 µL of hot 1× Laemmli buffer. Then 20 µL samples were run on a 4–15% SDS-PAGE and blotted onto Immobilon P PVDF (polyvinylidene fluoride) membrane (0.45 µm pore size, Millipore, (Budapest, Hungary). After blocking with 0.5% nonfat dry milk powder in PBS containing 0.1 % Tween 20, membranes were labeled 1:700 with HF1 alpha antibody (PA1-16601, ThermoFisher, (Waltham, MA, USA). As secondary antibodies goat anti-rabbit whole IgG antibodies were used (111-035-003, Jackson ImmunoResearch, (Cambridgeshire, UK)
Proteins were visualized using the ECL system (Luminata Forte Western HRP substrate, Millipore, Budapest, Hungary, # WBLUF0100).
Quantification was carried out using ImageJ software by referring the relative density of HF1 alpha bands to the untreated sample relative density. Data are shown as adjusted relative density by referring the relative intensity of HF1 alpha bands to the corresponding loading control intensities (Actin: Sigma-Aldrich, A2103 or GAPDH: Invitrogen, PA1-987).

Total protein extracts were prepared through extraction with RIPA buffer and proteins quantified using a bicinchoninic acid assay (Pierce Biotechnology, Rockford, IL, USA). For Western blot analyses, 30 μg of protein extracts were run on SDS-PAGE and transferred onto Immobilon P PVDF (Millipore, Billerica, MA, USA) or nitrocellulose (Bio-Rad, Hercules, CA, USA) membranes. Blots were incubated with antibodies specific for p53 (Ab-6; EMD Millipore, San Diego, CA, USA), phospho(S473)-AKT, AKT phospho(S235/236)-ribosomal protein S6, ribosomal protein S6 (Cell Signaling, Danvers, MA, USA), p62 (Enzo Biologicals, Plymouth Meeting, PA, USA), MitoProfile Total OXPHOS Cocktail (complex I-NDUFB8 subunit, CII-SDHB subunit, CIII-UQCRC2 subunit, and CIV-mitochondrial COX1 subunit) (Abcam, Cambridge, MA, USA), beta-actin (Sigma, St Louis, MO), Parkin (H-300, Santa Cruz Biotechnologies, Santa Cruz, CA, USA), TFAM (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), and voltage dependent anion channel (VDAC) (4866; Cell Signaling, Beverly, MA, USA) according to manufacturer’s instructions.