Clarity western ecl kit

PBMC lysates left over after p62/NBR1 assay were used for Westernblot analysis. Samples containing 10 µg of proteins were separated by gel electrophoresis and transferred to nitrocellulose membranes as described before [27 (link)]. The membranes were blocked overnight in 1 x Tris-buffered saline (TBS) containing 0.1 % Tween-20 and 3% bovine serum albumin at room temperature. The membranes were washed extensively with 1×TBS buffer and then probed with anti-LC3 or anti-p62 polyclonal antibody followed by horseradish peroxide-conjugated secondary antibody (goat anti-rabbit). After additional rinsing with 1×TBS buffer containing 0.1 % Tween-20, the membranes were exposed to a chemiluminescent substrate in the presence of hydrogen peroxide, using Clarity Western ECLkit (BioRad). A VersaDoc Model 4000 (BioRad) imaging system was used to capture the image.

Protein extracts were prepared from heads or dissected brains. Cell homogenization was done in RIPA buffer (150 mM sodium chloride, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 50 mM Tris, pH 8.0, supplemented with protease inhibitor cocktail from Roche Life Science). After 2 hrs of lysis at room temperature, Laemmli buffer was added before to boil the samples during 5 min.
Samples were analyzed by SDS-PAGE. To separate alpha and beta-tubulins, we used a protocol described by Banerjee and collaborators^{63 (link)}. Separated proteins were electrophoretically transferred onto nitrocellulose membrane (Hybond C-Extra, Amersham Biosciences) prior to blotting. Primary and secondary antibodies were incubated in 5% milk in PTX (PBS, 0.1% TritonX100) and washes were done with PTX. Immunodetection was done with Clarity Western ECL kit (BIO-RAD). Chemiluminescence detection was acquired with ChemiDoc Touch Imaging System (BIO-RAD). The following primary antibodies were used: mouse GT335 (1/200, AdipoGen), mouse 1D5 (1/500, Synaptic System), mouse anti-acetylated-tubulin (1/2000, Sigma), mouse DM1A (1/4000, anti-alpha tubulin from Sigma), mouse E7 (1/1000, anti-beta tubulin from DSHB), mouse anti-actin (1/2000, ThermoScientific). Secondary antibody used was HRP-linked goat anti-mouse (1/10000, Jackson ImmunoResearch).

Protein extracts from renal tissues were prepared by homogenization with glass tissue grinders (Kimble Chase, Rockwood, TN) in 1X cell lysis buffer (Cell Signaling, Danvers, MA). Samples were mixed with 1X Laemmli sample buffer, boiled, separated on a SDS-PAGE gel, and then transferred to PVDF membranes. Membranes were blocked with 5% nonfat dry milk, and then incubated overnight at 4° C with primary antibodies. Membranes were washed with TBST buffer (50 mM Tris, pH 7.4, 0.15 NaCl, and 0.05% Tween 20) and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody. Protein bands were visualized using the Clarity Western ECL kit (Bio-Rad, Hercules, CA) in a ChemiDoc MP system (Bio-Rad). The primary antibodies used were: monoclonal mouse-anti-β-actin (GenScript, Piscataway, NJ), polyclonal goat-anti-mouse ICAM-1 (R&D Systems, Minneapolis, MN), polyclonal rabbit-anti-rat ERK1/2 and polyclonal rabbit-anti-human phospho-ERK1/2 (Thr202/Tyr204) (Cell Signaling), polyclonal sheep-anti-mouse PC (Haematologic Technologies, Essex Junction, VT), and polyclonal rabbit-anti-mouse PAI-1 (ABCAM). The HRP-conjugated secondary antibodies used were: horse-anti-mouse IgG and goat-anti-rabbit IgG (Cell Signaling), donkey-anti-goat IgG (Jackson ImmunoResearch), and donkey-anti-sheep IgG (AbD Serotec, Raleigh, NC).

Kidneys were harvested as described above, flash frozen in liquid nitrogen, and stored at −80°C until use. They were homogenized in RIPA buffer (50 mM Tris‐HCl, 150 mM NaCl, 1% Nonidet P‐40, 0.1% SDS, 0.5% sodium deoxycholate, pH 7.4) containing PhosSTOP phosphatase inhibitor and cOmplete Mini protease inhibitor (both from Roche). Lysates were cleared by centrifugation (2 × 5 min at max speed), and total protein concentration was determined by bicinchoninic acid assay (Invitrogen). 80 µg protein per lane was loaded into SDS‐PAGE gels and transferred to PVDF membranes. Membranes were blocked with 5% milk (Carnation) in PBS with 0.05% Tween‐20 (PBST) and probed with primary antibodies for Smad2/3 (C‐8) (1:500, Santa Cruz Biotechnology #sc‐133098, RRID::AB_2193048) and CoxIV (1:20,000, Cell Signaling Technologies #4844, RRID:AB_2085427) overnight at 4°C. Membranes were then washed with PBST and probed with respective horseradish peroxidase‐conjugated secondary antibodies (GE Healthcare #NA931, RRID:AB_772210 and #NA934, RRID:AB_772206) at 1:2000 in blocking buffer for 1 hr at room temperature. Membranes were washed again with PBST and developed with Clarity Western ECL Kit (Bio‐Rad). Western blots were quantified using the Analyze Gels plugin on ImageJ.

Western blot analysis was performed as previously described else where (Wang et al., 2017b (link), 2018 (link)). Granulosa cells were collected after treatment for 48 h and lysed in RIPA buffer (catalog number: 89900; ThermoFisher, Rochford, IL, USA), then denatured by boiling for 5 min with bromophenol blue and frozen at −80 °C. The proteins were separated by 12% polyacrylamide gel electrophoresis, and then transferred to polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA). Firstly, the membrane were incubated with primary antibody: Bcl2 mouse monoclonal antibody (1:500, sc-7382; Santa Cruz, Dallas, TX, USA), Bax mouse monoclonal antibody (1:500, sc-20067; Santa Cruz, Dallas, TX, USA), caspase-3 rabbit polyclonal antibody (ab13847; Abcam, California, USA) and β-actin mouse monoclonal antibody (1:1000, SC-47778; Santa Cruz, Dallas, TX, USA). Later, the membrane was detected by incubation with HRP labeled goat anti-rabbit secondary antibody (SC-2054; Santa Cruz, Dallas, TX, USA) or goat anti-mouse secondary antibody (1:5000; SC-2005; Santa Cruz, Dallas, TX, USA), respectively. Finally, membranes was incubated with the Clarity Western ECL kit (catalog number: 170-5060; Bio-Rad Laboratories, Hercules, CA, USA), and scanned in a ChemiDocXRS chemiluminescent imaging system (Bio-Rad, Hercules, CA, USA).