Anti rabbit and anti mouse hrp conjugated secondary antibodies

THP-1 cells were differentiated into macrophages with PMA as described above, and MSeA (0, 5, 10 μM) was incubated in the presence or absence of LPS for 30 min. The cells were collected, washed in cold PBS and lysed in RIPA buffer (Sigma, St. Louis, MO, USA) containing protease inhibitors. Equal amounts of protein (50 μg) were subjected to SDS-PAGE, and Western blot was performed on the proteins transferred onto nitrocellulose membrane, as described earlier [39 (link)]. Primary antibodies against pIκBα, IκBα and Nrf2 (Cell Signaling, Danvers, MA) (1:1000 dilution) and β-actin (Proteintech, Chicago, IL, USA) (1:10,000 dilution) were reacted separately with the blot. The HRP-conjugated anti-rabbit and anti-mouse secondary antibodies (Cell Signaling, Danvers, MA, USA) were incubated at a dilution of 1:3000. Band expressions were developed using Pierce^TM ECL reagents (Thermo Scientific, Rockford, IL, USA).

Standard methodologies were used. Protein extracts were separated by 10% or 8% SDS-PAGE and transferred to a PVDF membrane. Membranes were incubated using the following specific antibodies, including mouse anti-puromycin (1:500, DSHB), mouse anti-Vinculin (1:2000, Merck), mouse anti-GAPDH (1:2000, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-APP (1:2000, Merck), rabbit anti-ADAM10 (1:500, Abcam, Cambridge, UK), mouse anti-sAPPα (1:500, IBL America, Minneapolis, MN, USA), rabbit anti-OCT3/4 (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-MAP2 (1:2000, Merck), mouse anti-Nestin (1:1000 Santa Cruz Biotechnology), mouse anti-SAP97 (1:1000, ENZO Life Sciences, Farmingdale, NY, USA) and rabbit anti-FMRP (1:1000, produced in house PZ1 [52 (link)]), HRP-conjugated anti-rabbit and anti-mouse secondary antibodies (1:5000, Cell Signaling Technology, Danvers, MA, USA). Proteins were revealed using an enhanced chemiluminescence kit (Bio-Rad, Hercules, CA, USA) and the imaging system LAS-4000 mini (GE Healthcare, Chicago, IL, USA). Quantification was performed using the IQ ImageQuant TL software (GE Healthcare). Detection of GAPDH, Vinculin, and Coomassie staining were used as normalizers. For all SDS-PAGE PageRuler™ Plus Prestained Protein Ladder (10 to 250 kDa, Thermo Fisher Scientific) was used.

Exponentially growing cells were pelleted, washed twice in cold PBS and lysed in 1× Gel sample buffer (50 mM Tris–HCl pH 6.8, 4% SDS, 10% glycerol, 1 mM EDTA, 0.01% Bromophenol Blue and 5% β-Mercaptoethanol, minus or plus 300 mM NaCl). SDS-PAGE and immunoblotting were carried out as described previously (29 (link),38 (link)). The following antibodies were used for immunoblotting: anti-actin 1:5000 (A-2066, Sigma), anti-RGC-32 as described in (15 (link)), anti-Pumilio 1 at 1:5000 (A300-201A, Bethyl Laboratories, Inc.), anti-Pumilio 2 at 1:2000 (A300-202A, Bethyl Laboratories, Inc.), anti-EBNA2 (PE2) at 1:200, anti-EBNA3A (exalpha) at 1:1000, anti-LMP1 (CS1–4) at 1/300, anti-MYC (9E10 culture supernatant) at 1/15. HRP conjugated anti-rabbit and anti-mouse secondary antibodies (Cell Signalling) were used at 1:2000 and HRP conjugated anti-goat secondary antibodies at 1:5000 (DAKO A/S, Denmark). Western blot quantification was carried out using Li-COR Image studio software using images captured using the Li-COR Odyssey Imaging system. Signals were adjusted for background and normalised to the signal for actin.

Proteins were separated by SDS-PAGE gel with a current of 30 mA and transferred onto PVDF membrane with a current of 300 mA. After blocking with 5% milk for 1 h, they were incubated overnight with the following primary antibodies: anti-MRE11 (1:1000, Abcam), anti-AKT (1:1000, CST); anti-pAKT (phosphor s473) (1:1000, CST), anti-PTOP (1:1000, Abcam), anti-cleaved caspase-3 (Asp175) (1:1000, CST), and anti-caspase-3 (1:1000, CST) at 4°C. Then, the membranes were washed three times with PBST and incubated with HRP-conjugated secondary antibodies (1:5000). After being washed by PBST, the protein bands were treated with ECL and detected with the Gel Doc EZ Imaging System (Bio-Rad, Berkeley, CA, United States).
β-actin was obtained from BD Biosciences, (San Jose, CA, United States); GAPDH, β-Tubulin, and HRP-conjugated anti-rabbit and anti-mouse secondary antibodies were obtained from Cell Signaling Technology.

Two cervical cell lines (HeLa and SiHa) were maintained in our laboratory. The cells were cultured in DMEM (Gibco) medium with 10% FBS (Gibco) at 37°C in an atmosphere of 5% CO₂. Rabbit polyclonal antibodies to TRIM29 and C-Myc were purchased from Abcam (USA). Purified Mouse Anti-E-Cadherin, N-cadherin and β-catenin were purchased from BD Biosciences (USA). Anti-rabbit and anti-mouse HRP-conjugated secondary antibodies were obtained from Cell Signaling Technology (USA).