Protein inhibitor cocktail

The cell samples were lysed with cold radio-immunoprecipitation (RIPA) butter (Beyotime Biotechnology, Haimen, China) supplemented with a protein inhibitor cocktail (Roche, Branford, CT, USA, and a phosphatase inhibitor cocktail. Protein concentrations were determined through the BCA method. Lysates were heated at 95 °C for 5 min or at 60 °C for 30 min and then loaded on 10% gels (Bio-Rad, Hercules, CA, USA) for SDS–polyacrylamide gel electrophoresis. After electrophoretic separation, the proteins were transferred onto 0.2 μm PVDF membranes, blocked with 5% nonfat milk, and incubated overnight at 4 °C with the primary antibodies targeting anti-Fpn (NBP1-21502, Novus, Centennial, CO, USA), anti-TfR1 (13113, Cell Signaling Technology, Danvers, MA, USA), anti-GPX4 (ab125066, Abcam, Waltham, MA, USA), anti-β-actin (AA128, Biotechnology, Shanghai, China), β-actin used as internal standards. Membranes were washed and incubated with HRP-conjugated secondary antibody (Abmart, Shanghai, China) at room temperature for 1 h. The antibody-labeled proteins were detected by chemiluminescence using Chemiluminescent HRP Substrate in an Amersham™ Imager 600 (GE Healthcare, Chicago, IL, USA). β-actin was used to normalize the protein expression by optical intensity analysis using the ImageJ software V1.52 a (Wayne Rasband National Institutes of Health, Bethesda, ML, USA).

Cerebella of P7 and P30 Cstb^−/− and control mice (n = 3 per genotype) were lysed with 50 mM Tris (pH 8.0), 0.5% Nonidet P-40, 10% glycerol, 0.1 mM EDTA, 250 mM NaCl, 0.1 mM Na₃VO₄, 50 mM NaF, 4 mM dithiothreitol (DTT), 1× Protein inhibitor cocktail (Roche, Basel, Switzerland) using Lysing Matrix D tubes (Qbiogene, Carlsbad, CA, USA) and FastPrep® FP120 Instrument (Qbiogene, Carlsbad, CA, USA). Lysed proteins (15 µg) were separated with Protean TGX precast gels (Bio-Rad Laboratories, Hercules, CA, USA) and transferred on the nitrocellulose membrane. The primary antibodies used were rabbit anti-rat GABRA6 (1∶1000) (Synaptic Systems, Göttingen, Germany) and mouse anti-rat β-tubulin (1∶10 000) (Sigma, St. Louis, MO, USA), and the secondary antibodies used were anti-rabbit-IRDye 800CW (1∶10 000) (LI-COR Biosciences, Lincoln, NE, USA) and anti-mouse-Alexa Fluor 680 (1∶10 000) (Invitrogen®, Life Technologies, Carlsbad, CA, USA). The bands were detected with Odyssey infrared reader (LI-COR Biosciences, Lincoln, NE, USA). Signal intensities were detected with Image Studio 3.1 (LI-COR Biosciences, Lincoln, NE, USA) and normalized to the intensity of β-tubulin.

Cells were collected and lysed in ice-cold RIPA buffer (150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS), 50 mM Tris-HCl at pH 8.0) with protein inhibitor cocktail (Roche Holding AG, Basel, Kanton Basel-Stadt, Switzerland) at 4 °C for 60 min. Cell debris was discarded by centrifugation at 12,000 g for 30 min at 4 °C, and protein concentration in suspension was quantified using a Bradford protein assay (Bio-Rad, Hercules, CA, USA). A total of 30 μg of proteins was separated using SDS-PAGE and, subsequently, transferred to a PVDF membrane (PERKin Elmer Life Sciences, Boston, MA, USA). Desired proteins were stained with appropriated 1st antibodies and HRP-conjugated 2nd antibodies. After staining, whole membranes were immersed in ECL reagent (Bio-Rad) and chemiluminescent intensity detected by a LAS-3000 imager (Fujifilm, Minato, Tokyo, Japan). Chemiluminescence of each protein was normalized with chemiluminescence of GAPDH, and the protein levels were presented as the intensity ratio to the untreated controls.

For Immunoblotting analysis, HCT116 cells were treated with 0.5% DMSO, TNP and analogs for 4 h, and then cellular proteins were prepared with lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 10 mM NaF, 50 mM Na₄P₂O₇, 1 X Protein inhibitor cocktail (Roche)). Total proteins were quantified by bicinchoninic acid assay (23225, Thermo Fisher Scientific). Total 20 μg proteins were resolved in 8% SDS–PAGE, and then Akt phosphorylation was probed with primary antibodies against, phospho–Akt (Thr308) (4056) and Akt (9272) (Cell Signalling) and HRP-conjugated secondary antibody. The relative band intensities of Akt phosphorylation to total Akt were quantified with Image J software.

Method was modified from Justice et al. (1995 (link)). Retinal extract was prepared by homogenizing four retinas in 0.2 ml of Buffer A (PBS, protein inhibitor cocktail (Roche), bromophenol blue tracking dye), and clearing insoluble parts by centrifugation. 20 μl of 10% Triton X-114 (648468, Calbiochem) was added to 180 μl of the retinal extract, mixed by gentle inversion and pre-warmed to 37°C for 5 min. The sample was centrifuged (300× g, 10 min) at 37–40°C leading to a separation of aqueous and Triton X-114 layers, with the later becoming blue-colored due to migration of bromophenol blue into the detergent phase. The upper aqueous layer was collected in a new test tube and mixed with 20 μl of 10% Triton X-114. The lower Triton X-114 layer was mixed with 0.2 ml of Buffer A, to equalize the compositions and volumes of the two fractions. Then, 0.8 ml of RIPA buffer was added to each fraction, and the epitope-tagged Gγ₁ was captured with anti-HA magnetic beads and analyzed by Western blotting, as described above.