Criterion tgx precast midi protein gel

CLL cells were lysed in RIPA lysis buffer (Cell Signaling Technology, Beverly, MA) containing phosphatase inhibitor cocktail 2 and protease inhibitor cocktail (Sigma-Aldrich, MO, USA). Extract from cell lysates were separated on 4%–15% Criterion™ TGX™ Precast Midi Protein Gel (Bio-Rad Laboratories) and transferred electrophoretically to nitrocellulose membrane (Bio-Rad Laboratories). The membranes were incubated with the designated antibodies and HRP-conjugated secondary antibodies according to the manufacturer’s instructions. Bands were detected using MyECL Imager (Thermo Scientific, Rockford, IL).

For immunoblot analysis, cells were counted and washed twice with cold PBS prior to collection. 200,000 cells were lysed in 40 μL 1× Laemmli protein loading buffer and boiled at 95 °C for 5 minutes. Whole-cell lysates were run on a 4%–15% TGX precast protein gels or 4–15% Criterion™ TGX™ Precast Midi Protein Gel (Biorad,4561085, 5671083, 5671084) and transferred to a nitrocellulose membrane (Biorad, 1620115). Membranes were blocked with 5% milk for 1 hour prior to incubation with antibodies against CNOT3 (Abnova, H00004849-M01, 1:1000), c-MYC [NEB, 5605 S, 1:1000], p21 [CST, 2947 S, 1:1000], ACTIN (Sigma Aldrich, A3854, 1:5000). After overnight primary antibody incubation, the membranes were washed with 1 × PBST and then incubated with HRP-linked secondary antibodies (goat anti-mouse IgG [NEB, 7076 V] or goat anti-rabbit IgG [NEB, 7074 V], 1:3000) for 1 hour at room temperature. Immobilon ECL Western HRP Substrate (Millipore, WBKLS0500) and ECL Western Blotting Detection Reagent (Sigma, GERPN2105) were used to detect the protein bands on the Bio-Rad ChemiDoc Imaging System with chemiluminescence detection. The relative protein expression of each protein band was analyzed by Image J software, using β-actin as the reference. Uncropped and unprocessed scans of the most important blots were included in the Source Data file.

The tissues were homogenized in RIPA buffer, mixed with 0.1% SDS, applied to 10% Criterion™ TGX™ Precast Midi Protein Gel (BioRad, Hercules, CA, USA) for separation (90 V for 20 min and 220 V for 30 min) and electroblotted onto PVDF membranes (2.5 A, 25 V, 10 min) by using Trans-BlotTurbo Transfer System RTA Transfer Kits (BioRad, Hercules, CA, USA). The blots were incubated with 5% skimmed milk in TBST (0.05 M Tris-HCl, pH 7.4, 0.9% sodium chloride, and 0.1% Triton) to block unspecific staining followed by incubation with different primary antibodies in 3% skimmed milk in TBST overnight at 4 °C. After rinsing with TBST 3 × 10 min, the blots were incubated in secondary antibody conjugated with horseradish peroxidase for 1 h at room temperature. After rinsing with TBST 3 × 10 min, the signals of the blots were captured by using ChemiDoc Imaging Systems (BioRad, Hercules, CA, USA). To compensate for loading errors, the values were normalized to GAPDH staining of the same blots. For quantitation of the relevant bands, squares were made of the exact same size for each lane in Image Lab 6.0 (BioRad, Hercules, CA, USA), and the signal intensity was measured. The background was determined and eliminated by using the same square size outside the relevant protein band.

Protein levels were measured using western blot. Animal tissues were homogenized in RIPA buffer (Thermo Fisher Scientific) containing Halt Protease Inhibitor Cocktail (Life Technologies). Protein concentrations were determined using the BioRad DC protein assay, and protein was loaded at 40 μg onto a 4%–15% Criterion™ TGX™ Precast Midi Protein Gel (BioRad). Western blot membranes were probed with primary anti-eRF1 antibody (ab31799; Abcam) or anti-eRF3a antibody (ab49878; Abcam). An antibody against β-actin (A5316; Sigma) was used as a loading control. Membranes were then incubated with IRDye secondary antibodies (Li-COR) and scanned using an Odyssey infrared system (Li-COR). Images were quantified using Image Studio (Li-COR).
hFIX protein level in mouse plasma was measured by enzyme-linked immunosorbent assay (ELISA) using Human Factor IX (FIX) ELISA Kit (ab188393; Abcam) following manufacture instructions.

Protein lysate of pellet cultures was mixed with lammelli buffer, then heated for 5 min at 95 °C, cooled down and vortexed before loading to gel (4–20% Criterion TGX Precast Midi Protein Gel, Bio-Rad Laboratories, Hercules, CA, USA). We loaded 15μg of protein per well. Gels were run in 1x Tris/Glycine/SDS running buffer for 1h at 80V and protein transfer for 2h at 0.45A using a transfer system (Bio-Rad Laboratories, USA). Following the transfer, the nitro cellulose membrane was rinsed with Tris Buffered Saline with Tween 20 (TBST) (0.05% Tween) and blocked with TBST (0.5% Tween 20) + 5% dry milk (or BSA) in for 60 min. The membranes were incubated with primary antibodies towards described above (SOD2, vimentin, MGST1 and SYF2). HRP-conjugated secondary antibodies were used to detect the proteins with Clarity Western ECL Substrate 1:1 (Bio-Rad Laboratories, USA), and chemiluminescence signal was measured using Image Quant LAS 4000 CCD imager and software (GE Healthcare, Life Sciences, Chicago, IL, USA).

Patient-derived tumor cells were treated with cell lysis buffer (Cell Signaling Technologies, Danvers, Massachusetts, 9803S), complete mini protease inhibitor mixture tablets (Roche, 11836153001), and PhosSTOP Phosphatase Inhibitor Cocktail Tablets (Roche, 4906845001). Lysates were spun at 8,000 xg for 10 minutes at 4°C, mixed 3:1 with 4x Laemmli Sample Buffer (Bio-Rad, 1610747) with b-ME, and heated at 95°C for 5 minutes. Lysates were run on Criterion TGX Precast Midi Protein Gel (Bio-Rad, 5671083), transferred to a polyvinylidene difluoride membrane (Bio-Rad, 1704157), and blocked for 1 hour in TBS-T with 5% milk. Blots were probed overnight at 4°C with PERK (C33E10) Rabbit, Phospho-p44/42 MAPK (Erk1/2) Rabbit, Phospho-Akt (Ser473) (D9E) XP Rabbit, Akt (pan) (C67E7) Rabbit, or a-Tubulin (DM1A) Mouse (Cell Signaling Technology). HRP conjugated secondary antibodies, anti-rabbit, or anti-mouse IgG were used for rabbit and mouse primary antibodies, respectively. Blots were developed using Clarity^TM or Clarity Max^TM Western ECL Substrate (Bio-Rad, 1705060 and 1705062) and imaged using a Bio-Rad ChemiDoc touch MP Imaging System. For all western blots the same blot was probed repeatedly.