Hybond ecl nitrocellulose membrane

Transfected HeLa p62 KO cells were harvested in 50 mM Tris, pH 7.4, 2% SDS, and 1% glycerol. Cell lysates were cleared by centrifugation, and supernatants resolved by SDS-PAGE and transferred to Hybond-ECL nitrocellulose membrane (GE Healthcare). The membrane was blocked with 5% nonfat dry milk in PBS-T, incubated with primary antibody overnight, and HRP-conjugated secondary antibody for 1 h at room temperature. Proteins were detected by immunoblotting with a chemiluminescence Luminol kit (SC-2048, Santa Cruz Biotechnology) using a LumiAnalyst Imager (Roche Applied Sciences).

MCF-7 cells were seeded in Petri dishes in steroid-free culture condition. After 24 hours of treatment with indicated compounds cells were washed with PBS before lysis in RIPA buffer: TBS containing 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 50 mM NaF, 0.1 mM sodium orthovanadate, 0.6 mM PMSF, 0.3 mM TPCK, and protease/phosphatase inhibitor mix (Fisher Scientific). Lysates were clarified and protein concentration of each sample was determined by BCA assay (Pierce). After addition of LDS Sample 4x buffer (Invitrogen), samples were boiled for 5 minutes and submitted to electrophoresis on a 4–12% SDS-PAGE gel (Invitrogen). Separated proteins were electrotransferred onto Hybond ECL nitrocellulose membrane (GE Healthcare) using a semidry blotting apparatus (BioRad). Nonspecific sites were blocked with 5% nonfat dry milk in TBS containing 0.05% Tween 20 (2 hours, room temperature). Membranes were then incubated overnight at 4°C with primary antibodies (F-10 at 1/750 and MAB1501 at 1/3000 from Santa Cruz Biotechnology and Millipore, resp.). Detection was performed by chemiluminescence, using a peroxidase-coupled secondary antibody (1.5 hours, room temperature) and Western Pico Detection system (both from Pierce). Immunoblots were visualized using a ChemiDoc XRS+ camera and densitometric analyses were performed using ImageLab software (BioRad).

H7 flagellin was separated through a 12% SDS-PAGE as mentioned above and transferred onto Hybond ECL nitrocellulose membrane (GE Healthcare Life Sciences, Marlborough, MA, USA) using a Trans-Blot electrophoretic transfer cell (Bio-Rad). After membrane blocking with 5% (w/v) milk powder (Sigma) in PBS at 4°C overnight, it was sequentially incubated first with 1/2000 rabbit polyclonal anti-H7 flagellin (Abcam, Boston, MA, USA), 1/50000 plasma from H7-immunized BALB/c mice or 1/10000 plasma from control BALB/c mice, and second with 1/3000 HRP-conjugated goat polyclonal anti-rabbit IgG or anti-mouse IgG (Invitrogen, San Diego, CA, USA), all diluted in PBS containing 5% (w/v) milk powder. Incubations were carried out at room temperature for at least 1 h on a platform shaker and were washed 3 times during 5 min with 0.1% (v/v) Tween20 in PBS before and after each antibody step. Specific bands were visualized by enhanced chemiluminescence (ECL) method (GE Healthcare Life Sciences).

Whole-cell extracts were prepared in ice-cold lysing buffer (1 ml PBS) was added with 10 µl Triton X-100, 10 µl SDS 10%, 5 µl DTT 1 M, 6 µl PMSF 0.1%, 10 µl aprotinin). Total proteins (50 µg) were separated by electrophoresis in 10% denaturing SDS/polyacrylamide gel, then transferred to Hybond ECL nitrocellulose membrane (GE Healthcare Europe, Milan, Italy). After saturation of non-specific binding sites with 5% non-fat milk in Tris-buffered saline (TBS) 1x-Tween 20 0.05%, the membrane was immunoblotted overnight at 4°C with the primary antibody against COX-2 (1∶250) and subsequently probed with an anti-goat secondary antibody (1∶1000) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) overnight at 4°C. The membrane was stripped (Restore Western Blot Stripping buffer, Pierce Biotechnology, Rockford, IL, USA) and re-immunoblotted with anti-actin primary antibody (1∶10000) and then with anti-rabbit secondary antibodies (1∶7500) (Santa Cruz Biotechnology Inc.). The immunoreactive bands were visualized through enhanced chemiluminescence using the ECL-plus kit (GE Healthcare Europe) following the manufacturer's protocol. The bands were quantified by densitometric analysis using Image J 1.43 software. The results were evaluated as relative units determined by normalization of the density of each band to that of the corresponding actin protein band.

Protein lysates were prepared as previously described^{49 (link)}. Protein content was quantified by Bradford assay, and samples were separated in 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were electrophoretically transferred onto Hybond ECL nitrocellulose membrane (GE Healthcare Bio-Sciences Corporation, Piscataway, NJ, USA), and incubated with primary antibodies raised against human TrkA (sc-118, 1:1000), TrkB (sc-20542, sc-12, 1:1000), TrkC (sc-14025, sc-117, 1:1000), p75NTR (sc-8317, 1:1000), phospho-Trk (Tyr680/Tyr681, sc-7996-R, 1:1000), Erk1 (sc-94, 1:1000), phospho-Erk (sc-7383, 1:1000), α-tubulin (sc-8035, 1:1000; all from Santa Cruz Biotechnologies Inc., Santa Cruz, CA, USA), Akt (9272, 1:2000), and phospho-Akt (4060, 1:2000, all from Cell Signaling Technology, Danvers, MA, USA). Species-specific infrared secondary antibodies (Li-Cor Biosciences, Lincoln, NE) were used to detect bound antibodies, followed by visualization using Li-Cor Odyssey Infrared Imaging System. Densitometry was performed using Image Studio Lite software (Li-Cor Biosciences).

Milli-Q water was used for all solutions. Phosphate-buffered saline (PBS) was from EuroClone. Bovine serum Albumin (BSA), methanol, CAPS, ZnSO₄ and the anti-protease inhibitor cocktail (Complete, Roche, Basel, Switzerland) were from SIGMA Chemical Co. (St. Louis, MO, USA); Hybond-ECL nitrocellulose membrane was from GE (Chalfont St Giles, Buckinghamshire, UK). NuPAGE^® SDS-PAGE Gel Electrophoresis System components (mini gels, running and loading buffers, molecular weight markers), SYPRO™ Ruby Protein Gel Stain and BCA protein assay were supplied by Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). Anti-tumor susceptibility gene 101 (TSG101) mouse monoclonal antibody (mAb) was purchased from Abcam (Cambridge, UK); anti-CD9 mouse monoclonal antibody was from Invitrogen (Thermo Fisher Scientific). Species-specific secondary peroxidase conjugated antibodies and ECL reagents were from Pierce (Thermo Fisher Scientific).