The IF analysis was performed by seeding the cells on the coverslips for 24 hrs followed by the treatment with either vehicle or Tea C-dots for 3 or 24 h. Upon treatment, cells were fixed in 4% paraformaldehyde for 15 min. IF images were taken based the methods we described previously39 (link), as well those described in the immunofluorescence general protocol (Cell Signaling Technology, Inc). Antibody used for F-actin was Alexa-Fluor-555-phalloidin (Molecular Probes Life technologies, 1:5000). Images were taken using Carl Zeiss Cell Observer SD confocal microscope. Primary antibodies used are: pAKT(S473) (Cell signaling), pYAP1(S127) (Cell signaling), SUMO1(Santa Cruz), SUMO2/3/4(Santa Cruz), ARF(Santa Cruz), YAP(Santa Cruz).
Sumo 1
Manufactured by Santa Cruz Biotechnology
Sourced in United States, Japan
SUMO-1 is a recombinant protein that functions as a small ubiquitin-like modifier. It is used in laboratory research applications.
Automatically generated - may contain errors
Lab products found in correlation
Sumo2 3, by Abcam (4 mentions)
N ethylmaleimide, by Merck Group (3 mentions)
β actin, by Merck Group (3 mentions)
Gapdh, by Santa Cruz Biotechnology (3 mentions)
Ubiquitin, by Santa Cruz Biotechnology (3 mentions)
Ripa buffer, by Beyotime (2 mentions)
Senp3, by Cell Signaling Technology (2 mentions)
Pias1, by Abcam (2 mentions)
E cadherin, by Abcam (2 mentions)
Immobilon p membrane, by Merck Group (2 mentions)
Flag tag (flag), by Merck Group (2 mentions)
Alexa fluor 555 phalloidin, by Thermo Fisher Scientific (1 mentions)
Cell observer sd confocal microscope, by Zeiss (1 mentions)
Pyap1 s127, by Cell Signaling Technology (1 mentions)
Sumo2 3 4, by Santa Cruz Biotechnology (1 mentions)
Pakt s473, by Cell Signaling Technology (1 mentions)
Sirt1, by Abcam (1 mentions)
Phospho gsk 3β, by Santa Cruz Biotechnology (1 mentions)
Phospho akt, by Santa Cruz Biotechnology (1 mentions)
α sma, by Santa Cruz Biotechnology (1 mentions)
18 protocols using sumo 1
1
Immunofluorescence Analysis of Cell Signaling
2
Protein Analysis of Cell Extracts
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Cell protein was extracted from CFs, CMs, and H9C2 cells under different conditions for analysis. The blots were probed with primary antibodies including α-SMA (Santa Cruz, USA), COLI /COLIII (Santa Cruz, TX, USA), SP1 (Santa Cruz, TX, USA), SIRT1 (Abcam, MA, USA), SUMO1 (Santa Cruz, TX, USA), Phospho-AKT (Santa Cruz, TX, USA), Phospho-GSK3β (Santa Cruz, TX, USA), β-actin (ZSGB-Biotech, Beijing, China) and GAPDH (ZSGB-Biotech, Beijing, China). The blots were probed with the secondary antibody (ZSGB-Biotech, Beijing, China). The bands were detected by an imaging instrument (LI-COR Biosciences, NE, USA).
3
Western Blot Analysis of Protein Modifications
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Total protein was extracted from cells as previously described. Cells were lysed in RIPA buffer (Beyotime Biotechnology; China) containing 1% protease inhibitors (Roche Diagnostics, Mannheim, Germany) or 20 mM N-ethylmaleimide as appropriate (Sigma-Aldrich; St. Louis, MO, U.S.). The lysates were separated on an 8% or 12% SDS-PAGE gel and then transferred to nitrocellulose filter membranes. After being blocked in 5% non-fat milk, the membranes were incubated with the following primary antibodies: anti-PML (1:500 dilution; MBL, Nagoya, Japan), SUMO-1 (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, U.S.), SUMO-2/3 (1:500 dilution; Abcam, Cambridge, U.K.), UBC9 (1:500 dilution; Cell Signaling Technology, Beverly, MA, U.S.); RNF4 (1:500 dilution; Abcam, Cambridge, U.K.); anti-TGF-β1 (1:500 dilution; Cell Signaling Technology, Beverly, MA, U.S.) and anti-HERG (1:500 dilution; Alomone labs, Jerusalem, Israel). GAPDH (1:10,000 dilution; Research Diagnostics, Concord, MA, U.S.). Goat anti-rabbit (1:10,000 dilution; Alexa Fluor 700 conjugated; Molecular Probes/Life Technologies) served as the secondary antibody. Immunoblots were imaged using an LI-CORE Imaging System (LI-COR Biosciences, Lincoln, NE, U.S.) and Odyssey software was used for quantification of bands normalized to GAPDH.
4
Quantitative Western Blot Analysis of SUMOylation Pathway
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Samples from each age point were subjected to SDS-PAGE on 8–15% polyacrylamide gels, depending on the protein being detected. For SUMO blots, gradient gels (4–20%) were used. Separated proteins were transferred to nitrocellulose membrane and immunoblotted. The primary antibodies used were: β-III-tubulin (1:5000, T8660), β-actin (1:2500, A5441), Syntaxin1A (1:5000, S0664) all from Sigma-Aldrich. PSD95 (1:1000, AB1596), GluA1 (1:1000, AB2263), NMADR1 (1:1000, AB9864) from Millipore. Aos1 (1:200, sc-46766), Uba2 (1:200, sc-376305), SUMO-1 (1:500, sc-5308) from Santa Cruz Biotechnology. Ubc9 (1:250, 610749) from BD Biosciences; PIAS1 (1:2000, 77231) from ABCAM; PIAS3 (1:1000, AP1245a) from Abgent; SENP3 (1:4000, D20A10) Cell Signaling Technology; SUMO-2/3 (1:100, Clone 8A2, produced in house) from DRHB. Luminescent or fluorescent signal was captured with a Li-COR Odyssey Fc system and quantified using Image Studio 2.1 provided by Li-COR. For SUMO quantification the whole lane was selected and quantified as well as individual bands with strong signal.
5
Immunoblot Analysis of Cellular Proteins
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Lysate, IP or His‐PD Samples were resolved by SDS–PAGE (10–15% gels) and transferred to Immobilon‐P membranes (Millipore Inc.), which were then immunoblotted with the following antibodies against: β‐actin (Sigma), CHIP (Cell Signaling), Drp1 (Cell Signaling), Fis1 (Proteintech), Flag (Proteintech), FTH1 (Cell Signaling), GAPDH (Santa Cruz biotechnology), GST (GE Healthcare), LC3 (Cell Signaling), p62 (Cell Signaling), SENP3 (Cell Signaling), SENP5 (Proteintech), SUMO‐1 (Santa Cruz biotechnology), SUMO‐2/3 (Cell Signaling; MBL) or Tom20 (Santa Cruz biotechnology). Immune complexes were detected using either HRP‐conjugated secondary antibodies (Sigma) followed by enhanced chemiluminescence (GE Healthcare) or using fluorescent secondary antibodies (LI‐COR).
Each immunoblot presented is representative of at least three experiments carried out using different cell populations.
Each immunoblot presented is representative of at least three experiments carried out using different cell populations.
6
Western Blot Analysis of EMT and TGF-β Signaling
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Whole-cell lysates were extracted by Radio Immunoprecipitation Assay (RIPA) lysis buffer (Beyotime, China) with protease (Roche, USA). Phosphatase inhibitors (Roche, USA) were added for primary antibodies against p-SMAD2/3. Protein samples of 70 μg per lane were loaded onto 10% SDS-PAGE gels and transferred onto nitrocellulose membranes (PALL, Germany) for 90 min. For immunodetection, the membranes were incubated with primary antibodies at 4°C overnight: E-cadherin (ab76055, Abcam, Cambridge, UK) at 1:1,000 dilution, α-catenin (ab52227, Abcam, Cambridge, UK) at 1:10,000 dilution, Vimentin (HPA001762, Sigma-Aldrich, USA) at 1:250 dilution, N-cadherin (C3865, Sigma-Aldrich, USA) at 1:500 dilution, SMAD4 (sc-7966, Santa Cruz, USA) at 1:200 dilution, p-SMAD2/3 (sc-11769, Santa Cruz, CA, USA) at 1:200 dilution, or SMAD2/3 (sc-133098, Santa Cruz, CA, USA) at 1:200 dilution, SUMO-1 (#sc-9060; Santa Cruz Biotechnology, CA, USA) at a 1:200 dilution, or SUMO-2/3 (#ab3742; Abcam, Cambridge, MA, USA), at a 1:500 dilution. After incubation with a secondary antibody and gentle shaking at room temperature for 30–60 min, the membranes were scanned using an Odyssey Infrared Imaging System (Li-COR, USA). All experiments were done at least five times. The values of protein band densities were normalized to those of the solution or blank control group.
7
Immunofluorescence Analysis of OSCC Cells
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Tca8113 and Cal-27 cells were plated onto a 24-well plate, and GA was dissolved in DMSO and diluted with RPMI 1640 medium, which was applied to OSCC cells 30 min before treatment with TGF-β1. The cells were cultured for 24 h in the presence of varying concentrations of GA (5 μM). The cells were washed with cold sterilized PBS for five times, fixed in 4% paraformaldehyde for 1 h, penetrated by 0.5% Triton X-100 (Sigma-Aldrich, MO, USA) for 15 min, and blocked with 0.1% bovine serum albumin (BSA) for 1 h. The cells were incubated at 4°C overnight with E-cadherin (ab76055, Abcam, Cambridge, UK) at 1:200 dilution, Vimentin (HPA001762, Sigma-Aldrich, MO, USA) at 1:200 dilution, SMAD4 (sc-7966, Santa Cruz, CA, USA) at 1:50 dilution, and SUMO-1 (1:50; #sc-9060; Santa Cruz Biotechnology, CA, USA). The samples were incubated with goat anti-mouse IgG (H+L) Highly cross-adsorbed secondary antibody, Alexa Fluor 488 (Invitrogen, Thermo Fisher, USA), or goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 594 (Invitrogen, Thermo Fisher, USA) at 1:200 dilution for 1 h. Nuclei were stained with DAPI (Beyotime, China) at room temperature for 5 min. The samples were visualized under a fluorescence microscope (Nikon 80i, Japan).
8
Detecting EAAT2 and Interacting Proteins
9
TFAP2A Regulation by SUMOylation
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Total protein was isolated 96 hours after siRNA transfection and 72-96 hours after SUMO inhibitor treatment using RIPA buffer with Halt Protease Inhibitor Cocktail (100X) (ThermoFisher Scientific, Rockford, IL, USA). Antibodies for TFAP2A (AbCam, Cambridge, MA, USA), PIAS1 (AbCam), CD44 (RD Systems, Minneapolis, MN, USA), Sumo 1 (AbCam), Sumo 2/3 (AbCam) and GAPDH (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) were used for Western blot analysis. Immunoprecipitations were performed using the Pierce Co-Immunoprecipitation Kit (Thermo Fisher) using antibodies for TFAP2A (AbCam) or Sumo 1 (AbCam) and Sumo 2/3 (AbCam) with rabbit IgG as a control.
10
Cell Culture Conditions for Glioma and HEK293 Cells
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HEK293 and HEK293T cell lines were purchased from American Type Culture Collection. Several glioma stem-like cell lines derived from both primary (GSC923) and recurrent (GSC827, GSC604) glioblastoma tumors and normal murine neural stem cells (MNSC) were described previously31 (link). All cells were grown at 37 °C in 5% CO2. HEK293 cells were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine and 1% penicillin-streptomycin (Gibco), and the GSC and MNSC were cultured in NBE complete media31 (link). Chemical such as MG132, N-ethylmaleimide, NMS-873, cyclohexamide and ATRA were purchased from Sigma-Aldrich. Puromycin was from Life Sciences. Antibodies used in Western blots and immunoprecipitations included: RARA, RXRA, Sumo1, Sumo2, Ub, VCP from Santa Cruz Biotechnology, DDK from Origene, β-actin from Sigma-Aldrich, normal rabbit IgG and normal mouse IgG from Dako, and Histone H3 from Cell Signaling.
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