Anti ha antibody

Cells were fixed with cold acetone for 10 min. After washing, cells were incubated in PBS containing 0.3% Triton X-100 for 10 min and blocked with 1% BSA for 30 min. Then, cells were incubated with anti-Myc antibody (MEDICAL & BIOLOGICAL LABORATORIES CO., LTD., mouse, 1:500 dilution), and anti-HA antibody (Proteintech, rabbit, 1:100 dilution) at 4°C overnight. Following additional washes, cells were incubated with the appropriate secondary antibody (1:200; Beyotime Biotechnology) at room temperature for 1 h, and then stained with DAPI. Images were acquired using Zeiss Laser scanning confocal microscope (Lsm880 NLO).

H1299 and U2OS cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum in an atmosphere of 5% CO₂ at 37 °C. For mammalian expression of p32 and p53, the corresponding cDNAs were amplified by PCR and ligated into the correct reading frames of pIRESneo (Clontech Laboratories, Inc., Mountain View, CA, USA) containing 5′ FLAG or hemagglutinin (HA) coding sequences. For bacterial expression of p32, p53, SRp30c, and p66α, their cDNAs were amplified by PCR and inserted into pET15b and/or pGEX‐4T1 vectors. To generate mutant p32 expression vectors, wild‐type p32 cDNA was mutated by using Q5® Site‐Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA, USA) after the construction. All constructs were verified by DNA sequencing. Further details of plasmid constructions are available upon request. Antibodies used in this study are as follows: anti‐β‐actin and anti‐FLAG antibodies from Sigma‐Aldrich, St. Louis, MO, USA; anti‐His antibody from LifeTein, Somerset, NJ, USA; anti‐lamin antibody from Active Motif, Carlsbad, CA, USA; anti‐HA antibody from Proteintech, Rosemont, IL, USA; and anti‐tubulin, anti‐p32, and anti‐p53 (DO‐1) antibodies from Santa Cruz Biotechnology, Dallas, TX, USA.

Chromatin immunoprecipitation (ChIP) assays were performed using a SimpleChIP Enzymatic Chromatin IP kit (Cell Signaling Technologies, Danvers, MA, USA). Cells were fixed with 1% formaldehyde for 10 min, and glycine with 0.125 M was added to stop the fixation. Chromatin was treated with micrococcal nuclease, sonicated, and immunoprecipitated with normal rabbit IgG (negative control) or anti-HA antibody (Proteintech, Rosemont, IL, USA) overnight at 4 °C. After the reverse cross-linking and DNA purification, immunoprecipitated DNA was quantified by qPCR.
Primer sequences were as follows:
CDK4#1-Forward: 5′-GGACCCAAGCAGACAGAGAG-3′
CDK4#1-Reverse: 5′-GGTGGGTGCTTTGTAAGCCT-3′
CDK4#E-box-Forward: 5′-GTGGCCTAGGTTGCCATGGCAC-3′
CDK4#E-box-Reverse: 5′-CTCACCATGTGACCAGCTGCC-3′

The cells were lysed by 1x Laemmli buffer containing protease inhibitors (0.5mM PMSF and 1x Complete Protease Inhibitor Cocktail; Roche) and phosphatase inhibitors (50mM NaF, 1.5mM Na3VO3). The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) before transferring to nitrocellulose membranes. SDS-PAGE was performed according to published protocol [21 ]. The following antibodies were used: anti-Flag (Sigma, 1:2000), anti-HA antibody (ProteinTech, 1:1000) and anti-beta actin (Sigma, 1:5000). Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Pierce. Chemiluminescent reagents (Pierce) were used to visualize the protein signals under the LAS-3000 system (Fujifilm).

Cultured cells and tumor tissues from nude mice were lysed with ice-cold RIPA buffer containing freshly added PMSF. Total protein was quantified using a BCA Protein Assay Kit (Beyotime, Guangzhou, China). Cell lysates  (40ug) were separated by SDS–PAGE and transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). Membranes were blocked in 5% bovine serum albumin (BSA) in 1× Tris-buffered saline containing 0.05% Tween 20 (TBST) for 1 h at room temperature and then incubated with primary antibodies overnight at 4°C, followed by incubation with HRP-conjugated secondary antibodies for 1.5 h at room temperature. The primary antibodies used were as follows: anti-PTPN1 antibody (201,974, 1:1000, Abcam), anti-HA antibody (51,064, 1:2000, Proteintech), anti-Src antibody (19,096, 1:2000, Proteintech), and anti-β-actin antibody (60,008–1, Proteintech). The secondary antibodies used were as follows: anti-rabbit (7404, CST) and anti-mouse (7076, CST). The bands were visualized using enhanced chemiluminescence (ECL) reagent (WBULS0500, Millipore) with the Tanon 5200 system (Tanon, Shanghai, China). The Human Phospho-Kinase Array (ARY003C, R&D Systems) was used according to the array procedure, and the intensities were analyzed by Image J.

HEK293 cells grown on coverslips (6x10⁴ cells/well in 24-well plate) were transfected with NgAgo expressing plasmid DNA with Fugene 6 or Lipofectamine 2000 (similar transfection efficiency in our hands). Twenty-four hours after transfection, the cells were fixed in 4% paraformaldehyde/PBS, pH7.4 at room temperature for 30 min, and permeablized with 0.1% Triton X-100/PBS at room temperature for 30 min. The cells were then blocked with 5% fat-free milk and incubated with anti-Flag antibody (Sigma, F3165, 1:2000, diluted in 1% milk in PBS) or anti-HA antibody (ProteinTech, # 51064-2-AP, 1:1000) at room temperature for 1 hour. After washing three times with PBS, the cells were stained with Alexa594 conjugated anti-mouse secondary antibody (for anti-Flag primary antibody) and anti-Rabbit secondary antibody (for anti-HA primary antibodies). Published immunostaining protocol was followed for immunostaining [23 (link)]. Cells were mounted in DAPI-containing mounting medium, and observed under an FV10i confocal microscope (Olympus Corporation, Tokyo, Japan). A 60x objective with a numerical aperture of 1.35 was used. The laser intensity and sensitivity parameters for each channel were kept constant for all samples in the same experiment.