Protein samples were prepared and then loaded onto an SDS-PAGE gel with electrophoretic transfer onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad), as previously described [24] (link). Antibodies against the following proteins were utilized: Beclin 1, cleaved caspase-3, γH2Ax, H2Ax, pCHK2, LC3, UVRAG, ATM (all from Cell Signaling Technology, 1∶1000), γ-tubulin, pATM (EPITOMICS, 1∶2000) and p62 (MBL, 1∶2000). Protein bands are quantified and normalized against γ-tubulin using ImageJ (National Institute of Health).
Uvrag
Manufactured by Cell Signaling Technology
Sourced in United States
UVRAG is a protein that plays a role in the regulation of autophagy, a cellular process that involves the degradation and recycling of cellular components. UVRAG is involved in the formation and maturation of autophagosomes, which are the vesicles that engulf and transport cellular materials to the lysosome for degradation.
Automatically generated - may contain errors
Lab products found in correlation
Beclin 1, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
P chk2, by Cell Signaling Technology (1 mentions)
γh2ax, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Lc3 2, by Cell Signaling Technology (1 mentions)
Ambra1, by Cell Signaling Technology (1 mentions)
Lc3 1, by Cell Signaling Technology (1 mentions)
Rpl26, by Cell Signaling Technology (1 mentions)
Nlrx1, by Cell Signaling Technology (1 mentions)
Vps34, by Cell Signaling Technology (1 mentions)
Hmgb1, by Cell Signaling Technology (1 mentions)
Dmi8 microscope, by Leica (1 mentions)
Goat anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Glomax 20 20, by Promega (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Anti goat or anti rabbit alkaline phosphatase conjugated antibody, by Promega (1 mentions)
6 protocols using uvrag
1
Western Blot Analysis of Autophagy Markers
2
Radiation-Induced Autophagy Regulation
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Fadu cells were treated with or without 4 Gy irradiation at a dose rate of 1.8 Gy/min in a linear accelerator (Primus Hi, Siemens Medical Instruments; Berlin, Germany). Twenty-four-hour later, cells were lysed in RIPA buffer (ThermoFisher Scientific) and lysates (20 µg) were analyzed on SDS-PAGE. Western blot analysis was performed as previously described.8 (link) The following antibodies were used: LC3B, P62, GAPDH, UVRAG (Cell Signaling Technology; Danvers, MA, USA).
3
Isolation and Characterization of Ginsenoside F2
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Ginsenoside F2 was isolated previously from leaves of Panax ginseng by a series of chromatographic procedures [9 (link)]. Ginsenoside F2 has a molecular mass of 784 Da and was isolated with 98% purity. Primary antibodies of PRR5, CISD2, Bcl-2L, NLRX1, RPS15, RPL26, p53, PUMA, Beclin-1, UVRAG, AMBRA-1, mTOR, LC3-II, LC3-I, and β-actin together with all secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The Atg5, Atg7, and Atg10 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
4
Spiral Sterol Saponin Autophagy Modulation
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The RCE-4 used in the present study was a spiral steranol saponin extracted and purified from Reineckia carnea [51 ]. The RCE-4 stock solution of 50 mmol·L−1 was prepared using DMSO and diluted to the desired concentrations in RPMI-1640 medium. Rabbit monoclonal antibodies against Beclin 1 (#3495S), Bcl-2 (#4223S), p-Bcl-2 (#2875T; #2827T), Mst 1 (#14946S), ATG 3 (#3415S), ATG 4B (#13507S), ATG 5 (#12994T), ATG 7 (#8558T), ATG 12 (#4180S), ATG 13 (#13468S), ATG 14 (#96752S), ATG 16L1 (#8089S), HMGB-1 (#MAB1690-SP), UVRAG (#NBP2-24482SS), Vps34 (#3358T), Rubicon (#8465S) and β-actin (#8457S) (Cell Signaling Technology, Boston, USA; R&D Systems, Minneapolis, MN, USA) were used at a dilution of 1:1000. HRP-conjugated secondary antibody (1:5000) was purchased fromSanta Cruz Biotechnology, Inc., Dallas, TX, USA.
5
Evaluating UVRAG Expression in HSCC
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Three matched HSCC tumor tissues from primary HSCC patients and recurrent HSCC patients who have only received radiotherapy were collected in the Department of Otolaryngology, Weihai Municipal Hospital. The use of human tissue samples was approved by the Research Ethics Committee of Shandong University and performed according to relevant guidelines and regulations. All patients provided informed consent, in accordance with the Declaration of Helsinki.
Paraffin-embedded hypopharyngeal squamous cell carcinoma samples were sectioned (4 μm) and mounted onto microscopic slides. Deparaffinized sections were incubated with the primary antibody at 4ºC overnight (UVRAG, Cell Signaling Technology), rinsed with PBS, and incubated with goat anti-rabbit secondary antibody (ZSGB-Bio; Beijing, China). Images were taken by a Leica DMi8 microscope (Leica Microsystems). Results of immunohistochemistry were quantified as follows: 0, no staining; 1, weak staining in <50% cells; 2, weak staining in ≥50% cells; 3, strong staining in <50% cells; and 4, strong staining in ≥50% cells.
Paraffin-embedded hypopharyngeal squamous cell carcinoma samples were sectioned (4 μm) and mounted onto microscopic slides. Deparaffinized sections were incubated with the primary antibody at 4ºC overnight (UVRAG, Cell Signaling Technology), rinsed with PBS, and incubated with goat anti-rabbit secondary antibody (ZSGB-Bio; Beijing, China). Images were taken by a Leica DMi8 microscope (Leica Microsystems). Results of immunohistochemistry were quantified as follows: 0, no staining; 1, weak staining in <50% cells; 2, weak staining in ≥50% cells; 3, strong staining in <50% cells; and 4, strong staining in ≥50% cells.
6
Validating miR-1185 Targets UVRAG and KRIT1
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Western blot was performed as previously described [22] using the following primary antibodies: Caspase-3 and UVRAG (Cell Signaling Technology, Danvers, MA, USA) as well as KRIT1 and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA). Anti-goat or anti-rabbit alkaline phosphatase-conjugated antibody (Promega, Madison, WI, USA) was used as a secondary antibody.
Target prediction and Luciferase activity assay miR-1185 target genes were predicted with TargetScan 6.2 (http://www.targetscan.org/). The pmiR-RB-REPORT TM dual luciferase reporter vectors carrying the wild-type (WT) or mutated (MUT) 3'-UTR of miR-1185 target genes (UVRAG and KRIT1) were constructed (Ribio Co., Guangzhou, China). Human embryonic kidney 293 (HEK-293) cells (ATCC, Manassas, VA, USA) were co-transfected with 200 ng of recombinant plasmid, 50 nM of miR-1185 mimic and 100 nM of miR-1185 inhibitor. After 24 h of transfection, the luciferase activities were measured with a dual luciferase reporter assay kit (Promega, Madison, WI, USA) on a luminometer (GloMaxTM 20/20, Promega).
Target prediction and Luciferase activity assay miR-1185 target genes were predicted with TargetScan 6.2 (http://www.targetscan.org/). The pmiR-RB-REPORT TM dual luciferase reporter vectors carrying the wild-type (WT) or mutated (MUT) 3'-UTR of miR-1185 target genes (UVRAG and KRIT1) were constructed (Ribio Co., Guangzhou, China). Human embryonic kidney 293 (HEK-293) cells (ATCC, Manassas, VA, USA) were co-transfected with 200 ng of recombinant plasmid, 50 nM of miR-1185 mimic and 100 nM of miR-1185 inhibitor. After 24 h of transfection, the luciferase activities were measured with a dual luciferase reporter assay kit (Promega, Madison, WI, USA) on a luminometer (GloMaxTM 20/20, Promega).
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