Whole cells were lysed in cell lysate buffer (PMSF:RIPA=1:100, Beyotime, China), and protein concentrations were quantified with a BCA protein quantification kit (Beyotime, China). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked in fat-free milk and incubated with specific antibodies at 4 °C for 12 hours. The antibodies included anti-PHLPP2 (Abcam, USA), anti-CD133 (Proteintech, USA), anti-CD44 (CST, USA), anti-EPCAM (CST, USA), anti-Nrf2 (CST, USA), and anti-GAPDH (Hangzhou Xianzhi, China). Membranes were then incubated with secondary antibody (ZSGB-bio, China) and detected using a chemiluminescence system (Bio-Rad).
Anti phlpp2
Manufactured by Abcam
Sourced in United States
Anti-PHLPP2 is a laboratory reagent used for research purposes. It is an antibody that specifically recognizes and binds to the PHLPP2 protein. PHLPP2 is a phosphatase enzyme that plays a role in cellular signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Anti phospho akt s473, by Cell Signaling Technology (3 mentions)
Anti mtor, by Cell Signaling Technology (3 mentions)
Anti akt, by Cell Signaling Technology (3 mentions)
Anti phlpp1, by Abcam (2 mentions)
Anti phospho gsk3β ser9, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Anti phospho p70s6k, by Santa Cruz Biotechnology (2 mentions)
Anti phospho igf 1 receptor, by Cell Signaling Technology (2 mentions)
Anti phospho her 2, by Santa Cruz Biotechnology (2 mentions)
Anti protor1, by Santa Cruz Biotechnology (2 mentions)
Anti deptor, by Santa Cruz Biotechnology (2 mentions)
Anti phospho akt t308, by Santa Cruz Biotechnology (2 mentions)
Anti phospho 4ebp1, by Santa Cruz Biotechnology (2 mentions)
Anti myc tag, by Cell Signaling Technology (2 mentions)
Anti phospho bad, by Cell Signaling Technology (2 mentions)
Anti mtor, by Santa Cruz Biotechnology (2 mentions)
Anti pi3k p85α, by Santa Cruz Biotechnology (2 mentions)
Anti phospho pi3k, by Cell Signaling Technology (2 mentions)
Anti phospho foxo1 ser256, by Cell Signaling Technology (2 mentions)
Anti pdk1, by Santa Cruz Biotechnology (2 mentions)
7 protocols using anti phlpp2
1
Protein Expression Analysis in Cells
2
Subcellular Localization of ZDHHC22
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Cells were seeded on coverslips and then transfected with pCMV6-Entry, pCMV6-ZDHHC22, or pCMV6-ZDHHC22-(C111A). After 48 hours, cells were fixed with 4% paraformaldehyde for 20 min and then permeabilized with 0.5% Triton X-100 for 10 min. After blocking with blocking buffer, cells were incubated with primary antibodies overnight at 4°C and then incubated with secondary antibodies for 1 hour at 37°C. DAPI (Roche, Palo Alto, CA, USA) was used for DNA counterstaining. Photomicrographs were acquired with a confocal laser scanning microscope (Leica, Hilden, Germany). The following antibodies were used for immunofluorescence: anti-Flag (1:400, #14793, Cell Signaling Technology), anti-mTOR (1:200, #2983, Cell Signaling Technology), anti-phospho-AKT(S473) (1:200, #4060T, Cell Signaling Technology), anti-PHLPP2 (1:40, ab71973, Abcam), ER-Tracker Red (1:2000, C1041, Beyotime), CoraLite488 (1:200, SA00013-1, Proteintech), and CoraLite594 (1:200, SA00013-4, Proteintech).
3
Immunohistochemical Quantification of PHLPP2
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For IHC analysis, the tissue microarray (TMA) specimens were deparaffinized, rehydrated, subjected to antigen retrieval, and endogenous peroxidase was blocked with 3% hydrogen dioxide. Then, the samples were incubated with primary antibodies recognizing human PHLPP2 (anti-PHLPP2, Abcam, USA) at 4 °C for 12 hours, and secondary antibodies (ZSGB-bio, China) were applied for 30 min at room temperature. Staining intensity was classified as 0 (lack of staining), 1 (mild staining), 2 (moderate staining) or 3 (strong staining); the staining percentage was designated as 1 (<25%), 2 (25%-50%), 3 (51%-75%), or 4 (>75%). The final staining score was calculated by multiplying the color intensity and positive cell percentage, and scores ranged from 0 to 12. Scores ≤3 were regarded as low expression, while scores of 4-12 were regarded as high expression.
4
Immunofluorescence Analysis of PHLPP1 and PHLPP2
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Seventy-two h after gene transfection, Eca109 cells were fixed in 4 % paraformaldehyde for 15 min at the room temperature, washed three times in PBS, and incubated for 60 min at the room temperature in a blocking solution (Beyotime). The primary antibody (anti-PHLPP1 and anti-PHLPP2 from Abcam) in the blocking reagent was added and incubated overnight at 4 °C. The next day, the cells were washed with PBS for three times, and the fluorescent probe conjugated secondary antibody (Jackson ImmunoResearch, USA) was added to the cells. The cells were further incubated for 60 min at the room temperature and then washed three times in PBS. Cell nucleus was counterstained with DAPI (Beyotime). Photos were then taken using a Nikon Fluorescence microscope.
5
Quantifying Protein Expression in Tissues
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Tissue specimens and cells were homogenized, and the total cellular protein was extracted using the RIPA Lysis Buffer (Beyotime, Jiangsu, China), according to the manufacturer’s instructions. Protein concentration was measured by the BCA method (Beyotime). Western blot was performed using anti-phospho-AKT, anti-AKT, anti-GAPDH (Cell Signaling Technology, Danvers, MA, USA), anti-PHLPP1, and anti-PHLPP2 (Abcam, Cambridge, MA, USA) antibodies. The relative levels of protein expression were quantified by densitometric scanning (Image J Software, NIH, Bethesda, MD, USA), and the relative gray values of protein levels were calculated based on the band intensity of protein of interest divided by the band intensity of loading control.
6
Comprehensive Western Blot Analysis of PI3K/AKT Pathway
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Western blot was performed as previously described 32 (link) with the following primary antibodies: anti-mTOR (1:100, sc-517464, Santa Cruz), anti-mTOR (1:1,000, #2972, Cell Signaling Technology), anti-phospho-IGF-1 Receptor (1:1000, #3021, Cell Signaling Technology), anti-phospho-HER-2(1:100, sc-81507, Santa Cruz), anti-Protor1 (1:100, sc-390496, Santa Cruz), anti-DEPTOR (1:100, sc-398169, Santa Cruz), anti-PDK1 (1:200, sc-293160, Santa Cruz), anti-PI3K p85α (1:100, sc-1673, Santa Cruz), anti-phospho-PI3K (1:100, sc-12929, Santa Cruz), anti-phospho-PI3K (1:1000, #4228, Cell Signaling Technology), anti-AKT (1:1,000, #4691T, Cell Signaling Technology), anti-phospho-AKT(S473) (1:1,000, #4060T, Cell Signaling Technology), anti-PHLPP2 (1:1,000, ab71973, Abcam), anti-phospho-AKT (T308) (1:100, sc-271966, Santa Cruz), anti-phospho-BAD (1:1000, #5284, Cell Signaling Technology), anti-phospho- GSK3β(Ser9) (1:1,000, #9323, Cell Signaling Technology), anti-phospho-FoxO1(Ser256) (1:1000, #9461T, Cell Signaling Technology), anti-phospho-4EBP1 (1:200, sc-293124, Santa Cruz), anti-phospho-p70S6K (1:200, sc-8416, Santa Cruz), anti-GAPDH (1:1,000, sc-47724, Santa Cruz), and anti-Myc-Tag (1:1,000, #2276s, Cell Signaling Technology).
7
Comprehensive Western Blot Analysis
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Western blot was performed as previously described [27] with the following primary antibodies: anti-mTOR (1:100, sc-517464, Santa Cruz), anti-mTOR (1:1,000, #2972, Cell Signaling Technology), antiphospho-IGF-1 Receptor (1:1000, #3021, Cell Signaling Technology), anti-phospho-HER-2(1:100, sc-81507, Santa Cruz), anti-Protor1 (1:100, sc-390496, Santa Cruz), anti-DEPTOR (1:100, sc-398169, Santa Cruz), anti-PDK1 (1:200, sc-293160, Santa Cruz), anti-PI3K p85α (1:100, sc-1673, Santa Cruz), antiphospho-PI3K (1:100, sc-12929, Santa Cruz), anti-phospho-PI3K (1:1000, #4228, Cell Signaling Technology), anti-AKT (1:1,000, #4691T, Cell Signaling Technology), anti-phospho-Akt(S473) (1:1,000, #4060T, Cell Signaling Technology), anti-PHLPP2 (1:1,000, ab71973, Abcam), anti-phospho-Akt (T308) (1:100, sc-271966, Santa Cruz), anti-phospho-BAD (1:1000, #5284, Cell Signaling Technology), antiphospho-GSK3β(Ser9) (1:1,000, #9323, Cell Signaling Technology), anti-phospho-FoxO1(Ser256) (1:1000, #9461T, Cell Signaling Technology), anti-phospho-4EBP1 (1:200, sc-293124, Santa Cruz), antiphospho-p70S6K (1:200, sc-8416, Santa Cruz), anti-GAPDH (1:1,000, sc-47724, Santa Cruz), and antimyc-Tag (1:1,000, #2276s, Cell Signaling Technology).
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