Antibodies used in this study include β-actin (AC004, ABclonal), Flag (SG4110-16, Shanghai Genomics Technology), Myc (SG4110-18, Shanghai Genomics Technology), Smurf1(ab300408, Abcam), Smurf2(#12,024, Cell Signaling Technology (CST)), p62(#23,214, CST), p53(sc-126, Santa Cruz), Akt(#4691, CST), p-Akt(#4060, CST).
Smurf1
Manufactured by Abcam
Sourced in United States
Smurf1 is a protein that functions as a small ubiquitin-like modifier (SUMO) E3 ligase. It plays a role in the SUMOylation process, which is a post-translational modification that attaches SUMO proteins to target proteins, thereby regulating their activity, localization, or stability.
Automatically generated - may contain errors
Lab products found in correlation
P akt, by Abcam (2 mentions)
Gapdh, by Abcam (2 mentions)
Peroxidase conjugated secondary antibody, by ZSGB-BIO (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Smurf2, by Cell Signaling Technology (1 mentions)
β actin, by ABclonal (1 mentions)
Runx2, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
β actin, by Abcam (1 mentions)
P β catenin, by Abcam (1 mentions)
Ecl detection solution, by Merck Group (1 mentions)
Bca protein assay kit, by Transgene (1 mentions)
Gel imaging system, by Bio-Rad (1 mentions)
β catenin, by Abcam (1 mentions)
Gsk 3β, by Abcam (1 mentions)
Keap1, by Proteintech (1 mentions)
Immpresstmhrp anti rabbit igg, by Vector Laboratories (1 mentions)
Goat anti rabbit igg secondary antibody, by Boster Bio (1 mentions)
Bcl 2, by Abmart (1 mentions)
12 protocols using smurf1
1
Antibody-Based Protein Detection Protocol
2
Evaluating Mesenchymal Stem Cell Markers
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BMMSCs were harvested in RIPA lysis buffer (Beyotime Institute of Biotechnology) and whole-cell protein extracts were quantified by a BCA assay, separated on SDS-PAGE 8%-12%, and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). Antibodies included p16ink4 (1:800, Abbiotec, San Diego, USA), p21 (1:500, Abcam, Cambridge, England), p53 (1:500, Cell Signaling Technology, Boston, MA, USA), Runx2 (1:500, Abcam), ALP (1:800, Abcam), OSX (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), TCF3 (1:500, Abcam), Smurf1 (1:500, Abcam). In addition, stripped membranes were reprobed with β-actin (1:5000, Abcam) as loading control. Signal detection was performed using the ECL Kit after incubation with an anti-rabbit or anti-mouse IgG secondary antibody (1:5000, CoWin Bioscience Co., Beijing, China). The relative band intensities in the scanned images were analyzed with Image J software (National Institutes of Health, Maryland, USA). Experiments were performed in triplicate.
3
Western Blot Analysis of Protein Expression
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Cells were harvested and lysed in RIPA lysis buffer (Thermo Fisher Scientific, USA) for extracting total proteins. The protein concentrations were measured by a BCA protein assay kit (Transgen Biotech, China), separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose (NC) membranes. The membranes were then blocked by 10% nonfat milk and incubated with the corresponding antibody (CKIP-1, Akt, p-Akt, Gsk3β, β-catenin, p-β-catenin, Smurf1, and GAPDH; Abcam, Cambridge, MA, USA) diluted according to the instructions for 12 h at 4°C. After washing 3 times with PBS/0.1% Tween-20, membranes were incubated at room temperature for 2 h with horseradish peroxidase-conjugated secondary antibody (anti-mouse IgG and anti-rabbit IgG at 1:10000 dilution; Abcam). Protein expression levels were detected with ECL detection solution (Millipore, USA) and visualized with chemiluminescence detection system. The bands signal were analyzed using a gel imaging system (Bio-Rad).
4
Comprehensive Immunoblotting Antibody Panel
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Primary antibodies used against proteins were as follow: SMURF1 (Santa Cruz, sc-100616), SMURF1 (Abcam, ab57573), BiP/GRP78 (ABclonal, A0241); Actin (Sigma, A1978), p62 (MBL, PM045); LC3B (Sigma, L7543); Phospho-IRE1(S724) (ABclonal, AP0878); IRE1(ABclonal, A17940); Phospho-JNK1/2/3 (T183+T183+T221) (Abmart, T55541); JNK (ABclonal, A4867); Phospho-eIF2α (Ser51) (ABclonal, AP0692); eIF2α (ABclonal, A0764); XBP1 (ABclonal, A1731); ATF4 (Santa Cruz, sc-390063); CHOP (ABclonal, A6504); BCL-2 (ABmart, T40056); Flag M2 (Sigma, F3165); GFP-tag (Proteintech, 66002-1-lg); HA-tag (MBL, M180-3); Myc-Tag (Proteintech, 16286-1-AP); NRF2 (Proteintech, 16396-1-AP); KEAP1 (Proteintech, 10503-2-AP); Ubiquitin (MBL, D058-3); alpha Tubulin (Abcam, ab7291), Histone H2B (Santa Cruz, sc-515808); Caspase3 (Santa Cruz, sc-7272); Ki67 (Abcam, ab16667).
Secondary antibodies used were as follow: goat anti-mouse IgG secondary antibody (BOSTER, BA1050); goat anti-rabbit IgG secondary antibody (BOSTER, BA1054); Alexa Fluor® 555 goat anti-mouse IgG (Life Technologies, A21425); ImmPRESSTM HRP anti-Rabbit IgG (VECTOR, MP-7401); ImmPRESSTM HRP anti-Mouse IgG (VECTOR, MP-7402); Rabbit anti-mouse IgG (CST, 58802) and Mouse anti-Rabbit IgG (CST, 93702) were used to avoid interference of the IgG heavy chain.
Secondary antibodies used were as follow: goat anti-mouse IgG secondary antibody (BOSTER, BA1050); goat anti-rabbit IgG secondary antibody (BOSTER, BA1054); Alexa Fluor® 555 goat anti-mouse IgG (Life Technologies, A21425); ImmPRESSTM HRP anti-Rabbit IgG (VECTOR, MP-7401); ImmPRESSTM HRP anti-Mouse IgG (VECTOR, MP-7402); Rabbit anti-mouse IgG (CST, 58802) and Mouse anti-Rabbit IgG (CST, 93702) were used to avoid interference of the IgG heavy chain.
5
Analysis of RhoA Activation and Downstream Signaling
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Cell lysates were prepared as previously described17 (link). An equal amount of protein was incubated with glutathione S-transferase-rhotekin (Upstate Biotechnologies, Inc., Lake Placid, NY, USA) for 45 min at 4 °C to collect the active form of RhoA (GTP-RhoA). Immunoblotting was performed with antibodies against ARHGAP26 (Abcam, Cambridge, MA, USA), RhoA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), MMP2 (Abcam), MMP7 (Abcam), SMURF1 (Abcam), CBL (Abcam), NEDD4 (Abcam), MDM2 (Abcam), WWP1 (Abcam), VEGF (Affinity, Cincinnati, OH, USA), β-catenin (Cell Signaling Technology, Danvers, MA, USA), GAPDH (Cell Signaling Technology), and horseradish peroxidase-labeled goat anti-rabbit IgG (Beyotime Biotechnology, Shanghai, China). Proteins on membranes were detected using an ECL system (Amersham Biosciences, Piscataway, NJ, USA).
6
Analysis of Colonic Cancer Proteins
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Surgically removed colonic cancer tissues and adjacent normal tissues were collected from three patients at Peking University Third Hospital (Beijing, China) and used for Western blot analysis. The experiments were approved by the Ethics Committee of Peking University Third Hospital.
Immunohistochemical staining for specific protein expression was performed on mouse tissue sections. In brief, sections (4 mm thick) were deparaffinized with xylene, followed by rehydration in ethanol. Hydrogen peroxide (3%) was used to eliminate endogenous peroxidase. Sections were incubated overnight at 4°C with primary antibodies against Kindlin-2 (Sigma-Aldrich), 1:200; Smurf1 (Abcam), 1:100; and Smad1 (Bioss), 1:100. After extensive washing in PBS buffer, sections were incubated for 30 min with secondary antibodies (Dako). Immunostaining was examined with a BX51 microscope (Olympus), and images were photographed with a 40× objective.
Immunohistochemical staining for specific protein expression was performed on mouse tissue sections. In brief, sections (4 mm thick) were deparaffinized with xylene, followed by rehydration in ethanol. Hydrogen peroxide (3%) was used to eliminate endogenous peroxidase. Sections were incubated overnight at 4°C with primary antibodies against Kindlin-2 (Sigma-Aldrich), 1:200; Smurf1 (Abcam), 1:100; and Smad1 (Bioss), 1:100. After extensive washing in PBS buffer, sections were incubated for 30 min with secondary antibodies (Dako). Immunostaining was examined with a BX51 microscope (Olympus), and images were photographed with a 40× objective.
7
Western Blot Analysis of Protein Signaling
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Treated and control cells were collected and exposed to RIPA lysis buffer. Separation of the boiled proteins in sample buffer was achieved via SDS-PAGE. Proteins were then transferred to PVDF membranes, which were blocked using 5% nonfat dry milk followed by incubation (4°C) with primary antibodies. After 12 h, the second round of incubation at room temperature was implemented with peroxidase-conjugated secondary antibodies for 1 h. Finally, enhanced chemiluminescence was used to visualize bound antibodies.
Primary antibodies were used as per manufacturer’s instructions and standard dilutions: Smurf1 (ab57573, Abcam); Akt and p-Akt Ser473 (#9272 and #4060 respectively, CST); β-actin (A1978-200, Sigma); PTEN (sc-7974, Santa); PTEN (10047-1-AP, Proteintech); PTEN (ABM-2052, Cascade BioScience); EGFR (#4267, CST); Phosphotyrosine (p-4G10) (05-321X, EMD Millipore); HA (M180-3, MBL International); Ki67 (ab15580, Abcam); p-p70S6K Thr389 (#9205, CST); p70S6K (sc-8418, Santa); 4EBP1 (#9644, CST); p-4EBP1(#2855, CST). The following secondary antibodies were used: Goat anti-Rabbit IgG (H+L) (HRP) Polyclonal (PI-31460, Thermo Fisher); Goat anti-Mouse IgG (H+L) (HRP) Polyclonal (PI-31430, Thermo Fisher).
Primary antibodies were used as per manufacturer’s instructions and standard dilutions: Smurf1 (ab57573, Abcam); Akt and p-Akt Ser473 (#9272 and #4060 respectively, CST); β-actin (A1978-200, Sigma); PTEN (sc-7974, Santa); PTEN (10047-1-AP, Proteintech); PTEN (ABM-2052, Cascade BioScience); EGFR (#4267, CST); Phosphotyrosine (p-4G10) (05-321X, EMD Millipore); HA (M180-3, MBL International); Ki67 (ab15580, Abcam); p-p70S6K Thr389 (#9205, CST); p70S6K (sc-8418, Santa); 4EBP1 (#9644, CST); p-4EBP1(#2855, CST). The following secondary antibodies were used: Goat anti-Rabbit IgG (H+L) (HRP) Polyclonal (PI-31460, Thermo Fisher); Goat anti-Mouse IgG (H+L) (HRP) Polyclonal (PI-31430, Thermo Fisher).
8
Whole-cell Protein Extraction and Western Blotting
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Total cell lysates from Passage 0 BMSCs and iMACs were extracted using RIPA buffer (50mM Tris pH 7.5, 150mM NaCl, 1% Triton X, 1mM EDTA, 0.1% SDS, 0.5% Na-deoxycholate) supplemented with a protease inhibitor cocktail (Roche). For protein isolation from whole bone, joint and skeletal muscle, powderized specimens were resuspended in RIPA. Protein lysates (25–60μg) were separated by SDS-PAGE mini-protean gels (Bio-Rad) and transferred to nitrocellulose membranes. Membranes were probed using primary antibodies against Smurf2 (Abcam, 1:1000), Smurf1 (Abcam, 1:2000), Col2 (Millipore, 1:2000), Sox9 (Millipore, 1:1000), GAPDH (Sigma, 1:5000), tubulin (Sigma, 1:5000) and goat secondary antibodies against rabbit (1:5000) and mouse (1:10,000) IgG and then visualized using a chemiluminescent substrate (Pierce).
9
Western Blot Analysis of Cellular Proteins
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Cancer cells were dissociated in RIPA lysis buffer (P0013D) (Beyotime, Haimen, China) and PMSF (ST506) (Beyotime). Split products were centrifuged at 18 500 g , and the supernatants were collected. A Bradford Protein Assay Kit (P0006) (Beyotime) was used to analyze protein concentration, and the proteins were subject to 10% SDS/PAGE electrophoresis. After separation the proteins were transferred onto poly(vinylidene difluoride) membranes (Sigma‐Aldrich), and the membranes were blocked with 5% skim milk (Guangming, Shanghai, China) and incubated with primary antibody at 4 °C overnight. Then, specimens were hatched with secondary antibody conjugated with horseradish peroxidase (Cell Signaling Technology, Beverly, MA, USA). Signals were detected using a horseradish peroxidase chemiluminescent kit (Thermo Fisher Scientific) using an optional CCD camera and image processing system (Bio‐Rad, Hercules, CA, USA). GAPDH (G8140, US Biological, Swampscott, MA, USA) was used as a loading control. SMURF1, DAB2IP and p‐RSK1 (Ser380) were purchased from Abcam. p‐AKT (Ser473), AKT, p‐mTOR (Ser2448), mTOR, p‐ERK (Thr202/Tyr204), ERK and RSK1 were obtained from Cell Signaling Technology.
10
Molecular Mechanisms of FEZF1-AS1 in Cancer
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The RPMI‐1640 medium and DMEM were purchased from Gibco (USA); Lipofectamine 2000 was purchased from Invitrogen (USA); the MTT kit was purchased from Sigma (USA); the cell death detection kit was purchased from Dalian Meilun Biotechnology Co., Ltd.; the reverse transcription kit and SYBR Green qPCR Master Mix were purchased from Takara (Dalian); Transwell chambers and Matrigel were purchased from BD (USA); the dual luciferase reporter gene assay kit, TRIzol reagent, and BCA kit were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; the FEZF1‐AS1 small interfering RNA (si‐FEZF1‐AS1), small interfering RNA controls (si‐NC), miR‐1254 mimics, mimic controls (miR‐con) and miRNA inhibitor empty vector plasmid (pcDNA), FEZF1‐AS1 overexpression vector (pcDNA‐FEZF1‐AS1), wild‐type luciferase reporter gene vector (WT‐FEZF1‐AS1), and mutant luciferase reporter gene vector (MUT‐FEZF1‐AS1) were provided by Sangon Biotech (Shanghai) Co., Ltd.; Smurf1, E‐cadherin, Vimentin, N‐cadherin, AKT, p‐AKT, c‐Myc, ZEB1, and GAPDH antibodies and secondary antibodies were purchased from Abcam (USA).
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