Horseradish peroxidase linked anti mouse igg

To conduct this assay, 10 ng of GST-tagged recombinant phosphorylated human ERK2 (R&D Systems, 1230-KS) was incubated with and without the indicated DUSP5 proteins (0.5 nM final concentration) for 5–15 min, as indicated. The reactions were halted with 2x Laemmli sample buffer and subjected to SDS-PAGE. The proteins were transferred to polyvinylidene difluoride (PVDF) and immunoblotted using antibodies to pERK (Cell Signaling Tech., #9106) and total ERK (Cell Signaling Tech., #9102). Bound antibodies were visualized using horseradish peroxidase-linked anti-mouse IgG (Cell Signaling Tech, #7076S) and anti-rabbit IgG (Cell Signaling Tech, #7074S), respectively, and ECL reagents (Pierce, 34708) according to the manufacturer’s protocol.

After collecting the cell medium, proteins expressed in HEK293 cells were extracted using ice-cold 1× lysis buffer (125 mM Tris-HCl pH 6.8, 0.5% SDS). Proteins in the medium were precipitated using Tricholoroacetic acid (TCA). In brief, the medium was spun down at 1,200 × g to pellet the cell debris and the supernatant was centrifuged at 16,000 × g after adding TCA (25% v/v). The protein pellet was washed 2 times with ice-cold acetone, and then resolved in 2X loading buffer. Equal amounts of proteins were loaded and separated by 10–15% SDS-PAGE and wet-transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA). After blocking for 30 min in Tris-buffered saline with Tween-20 (TBST) containing 4% nonfat dry milk, the membranes were incubated overnight at 4°C with mouse anti-HA-tag (1:3000; Cell Signaling, Danvers, MA) antibody as a primary antibody. Horseradish peroxidase-linked anti-mouse IgG (1:5000; Cell Signaling) was used as a secondary antibody for an 1-h incubation at room temperature. After detecting signals with ECL plus reagents (GE Healthcare Biosciences, Pittsburgh, PA), proteins were visualized by exposure of the membranes to X-ray films (GE Healthcare Biosciences).

In brief, 4 μL of extracted protein samples were loaded on the wells of 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated protein on SDS-PAGE was stained using Coomassie brilliant blue (CBB). Anti-His antibodies (primary antibody, Novagen^®; EMD Chemical, Inc., Germany) and horse radish peroxidase-linked anti-mouse IgG (secondary antibody; Cell Signaling Technology, Inc.) were used for detection in a western blot (WB) analysis.

All the antibodies were from Cell Signaling Technology (Danvers, MA, USA): mTOR (cat #2983), phospho-mTOR (Ser2481, cat #2974), phospho-mTOR (Ser2448, cat #5536), Rictor (cat #9476), phospho-Rictor (Thr1135, cat #3806), Raptor (cat #2280), AKT (cat #4691), phospho-AKT (Ser473, cat #4060), phospho-AKT (Thr308, cat #13038), phospho-AKT (Thr450, cat #9267), GSK-3β (cat #9315), phospho-GSK-3β (Ser9, cat #9336), PTEN (cat #9559), β-actin (cat #4970), 4E-BP1 (cat #9644), phospho-4E-BP1 (Thr37/46, cat #2855), S6K1/p70S6K (cat #9202), phospho-S6K1/phospho-p70S6K (Thr389, cat #9234), Myc-Tag (cat #2276), horseradish peroxidase-linked anti-rabbit IgG (cat #7074), horseradish peroxidase-linked anti-mouse IgG (cat #7076). Lipofectamine LTX, plus reagent, culture medium were from Invitrogen (Carlsbad, CA, USA). Antibiotic–antimycotic, propedium iodide (PI), rapamycin, GSK3β inhibitor (SB212763), PI3K inhibitor (LY294002 and wortmannin) and other chemicals were from Sigma–Aldrich (St Louis, MO, USA). Protease and phosphatase inhibitor cocktails were from Calbiochem (San Diego, CA, USA). Cycle Test Plus kit was from BD Bioscience (East Rutherford, NJ, USA). Super Signal West Pico imaging system was from Thermo Scientific (Rockford, IL, USA).

We followed the methods of Sun et al. Briefly, total protein of cells was harvested using RIPA lysis buffer (Beyotime). Protein extracts were separated by using SDS-PAGE gels, followed by the transfer to a PVDF membrane. After blocking with 5% nonfat milk, the bands were incubated with indicated primary antibodies (Tsg101, Cell Signaling Technology; CD63, Abcam; CD9, Abcam; GM130, Abcam; NAMPT, Adipogen; P16, Abcam; MMP-3, Abcam; ADAMTS-4, Abcam; Aggrecan, Abcam; Collagen II Abcam; Sirt1, Abcam; Sirt3, Abcam; Sirt5, Abcam; β-actin, Abcam) overnight under 4°C. Then, the bands were incubated with horseradish peroxidase-linked anti-mouse IgG (Cell Signaling Technology) or anti-rabbit IgG (Abcam) secondary antibody for 1 h. The bands were exposed with enhanced chemiluminescence (ECL, Thermo Fisher Scientific).

For western blotting, the whole cell lysates from equally confluent cultures were prepared in proportionate volume of laemmli buffer and denatured at 95 °C followed by SDS-PAGE. The gel was transferred onto a nitrocellulose membrane, blocked with 5% milk prepared in 1× TBST. The membrane was then incubated with the appropriate antibody, washed and probed with horseradish-peroxidase-conjugated secondary antibody. Enhanced chemiluminescence was used to visualize the protein bands. Quantity One Software (version 4. 6. 3; Bio-Rad) was utilized to quantitate the levels of specific proteins, which were expressed after normalization with the protein loading control. The following antibodies were used: mouse anti-hnRNPK antibody (ab23644, Abcam), rabbit anti-HuR antibody (ab200342, Abcam) and rabbit anti-HOXC10 antibody (orb539862, Biobryt Biotechnology). Antibodies to detect hnRNPK, HuR, and HOXC10 were validated by immunoblotting with siRNA-transfected cell lysates. Antibodies against loading control proteins Cdk2 (Sc-163) and β-actin (Sc-97778) were obtained from Santa Cruz Biotechnology. Horseradish-peroxidase-linked anti-mouse IgG (Cat no. 7076, Cell Signaling Technology) and horseradish-peroxidase-linked anti-rabbit IgG (Cat no. 7074, Cell Signaling Technology) were used as secondary antibodies in immunoblotting.