Cells were treated with WX-554 and WX-037 at 1 or 10 times the half maximal growth inhibitory concentration (GI50) in DMSO, or 0.5% (v/v) DMSO alone, for 24 h. Western blots were prepared, probed with phospho-4EBP1 (Thr37/46) (#2855), phospho-p44/42 MAPK (Thr202/Tyr204) (#4370), phospho-AKT (Ser473) (#4060) or phospho-S6 ribosomal protein (Ser235/236) (#4858) monoclonal antibodies obtained from Cell Signalling Technology (New England BioLabs (UK) Ltd, Hertfordshire, UK) and developed as described previously [23 (link)]. Blots were then stripped (100 mM 2-mercaptoethanol, 2% (w/v) SDS and 62.5 mM Tris pH6.8 at 55 °C for 30 min) and re-probed with the respective total monoclonal antibody (4EBP1 (53H11) (#9644), p44/42 MAPK (ERK1/2) (#4695), AKT (pan) (C67E7) (#4691) or S6 ribosomal protein (5g10) (#2217)) obtained from Cell Signalling Technology (New England BioLabs (UK) Ltd, Hertfordshire, UK) and developed as described above.
Akt pan c67e7
Manufactured by Cell Signaling Technology
Sourced in United Kingdom, United States
Akt (pan) (C67E7) is a rabbit monoclonal antibody that recognizes all isoforms of the Akt protein. Akt is a serine/threonine protein kinase that plays a key role in cell signaling pathways involved in cell proliferation, growth, and survival. This antibody can be used to detect and study Akt in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Phospho akt ser473, by Cell Signaling Technology (5 mentions)
Phospho akt ser473 d9e, by Cell Signaling Technology (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
P44 42 mapk erk1 2, by Cell Signaling Technology (1 mentions)
Phospho 4e bp1 thr37 46, by Cell Signaling Technology (1 mentions)
Phospho p44 42 mapk thr202 tyr204, by Cell Signaling Technology (1 mentions)
Phospho s6 ribosomal protein ser235 236, by Cell Signaling Technology (1 mentions)
4ebp1 53h11, by Cell Signaling Technology (1 mentions)
S6 ribosomal protein 5g10, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Hvin 1, by Merck Group (1 mentions)
Immobilin western chemiluminescent hrp substrate, by Merck Group (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
P egfr, by Cell Signaling Technology (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail 3, by Thermo Fisher Scientific (1 mentions)
Anti cd44, by Cell Signaling Technology (1 mentions)
Phosphatase inhibitor cocktail 2, by Thermo Fisher Scientific (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
13 protocols using akt pan c67e7
1
Evaluating Signaling Pathway Responses
2
Quantifying Phospho-Akt Levels in Lung Tumors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein extracts were obtained from control lungs, ADC and PSC tumors. Total protein extracts (50 µg) from each sample were subjected to SDS-PAGE and transferred to nitrocellulose membranes (Amersham Biosciences, Arlington Heights, IL, USA). Membranes were blocked in PBS (Phosphate-buffered saline) containing 5% BSA (bovine serum albumin) and immunodetection was perform using antibodies against phospho-Akt (Ser473) (D9E) (Cell Signalling Technology, Danvers, MA, USA), Akt (pan) (C67E7) (Cell Signalling Technology, Danvers, MA, USA) and vinculin (hVIN-1) (Sigma-Aldrich, San Luis, MO, USA). In all cases, membranes were incubated with a horseradish peroxidase (HRP)-labeled secondary antibody and detected by luminography using Immobilin Western Chemiluminescent HRP Substrate (Millipore, Burlington, MA, USA).
3
Protein Extraction and Signaling Pathway Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein extraction from cells were isolated in RIPA lysis buffer (25 mM Tris-HCl (pH 7.4), 10% (v/v) glycerol, 150 mM NaCl, 2 mM EDTA, 1% (v/v) phosphatase inhibitor cocktail II (Thermo Scientific), and 1% (v/v) protease inhibitor cocktail III (Thermo Scientific)). Then protein concentrations was determined using a Thermo Scientific Pierce BCA Protein Assay Kit and denatured in 1× Laemmli’s gel loading buffer. Cellular protein was separated by SDS-PAGE and transferred to PVDF membranes, then probed with primary antibodies against SALL4, p-EGFR (Tyr1068), ERK1/2 (L34F12), p-ERK1/2 (Thr202/Tyr204), AKT (pan) (C67E7), p-AKT (Ser473), and β-actin (Cell Signaling). Signals were detected using a chemiluminescence kit (Thermo Scientific). The sections of human lung cancers were subjected to hematoxylin-eosin staining and immunohistochemical staining using a VECTASTAIN ABC Kit (rabbit/mouse IgG) with anti-SALL4 and anti-CD44 (Cell Signaling) antibodies.
4
Western Blot Analysis of Exosomal Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells or exosomes were lysed in the RIPA buffer. Protein concentration was measured using the bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, #A53226, Waltham, MA, USA). The loading protein was separated by SDS/PAGE gels and transferred to Immobilon-P (Millipore) membranes. After being blocked by 5% (w/v) BSA at room temperature for 1 h, the membranes were incubated with relevant primary antibodies and peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch). The images were acquired by enhanced chemiluminescence (Millipore, #P90719, Billerica, MA, USA) with a Bio-Rad ChemiDoc XRS+ imaging system. The primary antibodies are as follows: β-actin (Santa Cruz Biotechnology, #sc-47778, 1:2000), MMP-9 (Abcam, #ab38898, 1:1000), vimentin (Abcam, #ab73843, 1:1000), PTEN (Cell Signaling Technology, #9559, 1:1000), Akt (pan) (C67E7) (Cell Signaling Technology, #4691, 1:1000), Phospho-Akt (Ser473) (Cell Signaling Technology, #4075, 1:1000), CD81 (Novus Biologicals, #NB100-65805, 1:1000), and CD63 (Novus Biologicals, #NB100-77913, 1:1000).
5
Western Blot Analysis of Phosphorylated Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed with PBS and lysed in 500 μL of complete RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS, pH 7.4 ± 0.2) with protease inhibitor (complete mini, Roche Diagnostics) and phosphatase inhibitor (Life Technologies). Total protein concentration was determined using a BCA quantification assay kit (Life Technologies). Equal amounts of protein were loaded onto a 4–15% gel (Biorad) for electrophoresis and the gel was transferred onto a nitrocellulose membrane by electroblotting. Membranes were blocked with 5% BSA (Sigma) for 1 h. Primary antibodies used were phospho-WNK1 (p-WNK1), WNK1, phospho-Akt (Ser473), and Akt (pan) (C67E7), all purchased form Cell Signaling Technology. Solutions of primary antibodies were prepared at concentrations recommended by the manufacturer. Membranes were incubated with the primary antibody solutions at 4 °C overnight. After repeated washing, membranes were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h, followed by another round of repeated washing. Detection was carried out with an ECL chemiluminescence detection kit (GE Healthcare) using a FluorChem E imaging system.
6
Investigating AKT Signaling in Human Skeletal Myoblasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human Skeletal Myoblasts (#A11440 or #A12555), low glucose Dulbecco's Modified Eagle Medium (#11885092), Horse Serum (#26050070), Bovine Serum Albumin (#A9576), and Pierce A/G Magnetic Beads (#88802) were purchased from Thermofisher Scientific Antibodies for Total AKT1 (#2938), Total AKT2 (#2964), Total AKT3 (#14293), Total AKT (#4685), p‐AKT S473 (#4060), p‐AKT1 S473 (#9018), p‐AKT2 S474 (#8599), AKT (Pan) (C67E7) (rabbit monoclonal; #4691), AKT (Pan) (40D4) (mouse monoclonal; #2920), and Human Insulin‐like Growth Factor 1 (#8917) were purchased from Cell Signaling Technologies. Secondary antibodies Anti‐Rabbit IgG HRP‐Linked Antibody (PN#7074) and Anti‐Mouse IgG HRP‐Linked Antibody (PN#7076) were purchased from Cell Signaling Technologies. Human Insulin Solution (#I9278) was purchased from Sigma Aldrich ImageJ was downloaded from the National Institutes of Health website.
7
Western Blot Analysis of Protein Targets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total soluble proteins (100 μg) extracted from the samples were resolved on 10% sodium dodecyl sulphate–polyacrylamide gels and transferred electrophoretically to a polyvinylidene fluoride membrane. The blots were blocked with 5% skim milk, followed by incubation with antibodies specific for PTN (C‐19) [Santa Cruz (sc‐1394) goat polyclonal antibody], SREBP‐1c [Abcam (ab3259) mouse monoclonal antibody], FAS [Santa Cruz (sc‐55580) mouse monoclonal antibody], phospho‐Akt (pSer473) [Cell Signaling Technology (#9271) mouse Polyclonal antibody], Akt (pan) (C67E7) [Cell Signaling Technology (#4691) rabbit monoclonal antibody], mTORC1 (7C10) [Cell Signaling Technology (#2983) rabbit monoclonal antibody] and phospho‐mTORC1 (Ser2448) [Cell Signaling Technology (#5536) rabbit monoclonal antibody]. Actin (I‐19) [Santa Cruz (sc‐1616) goat polyclonal antibody] was used as a loading control. The blots were then incubated with peroxidase‐labelled rabbit, mouse and goat secondary antibodies (1:2000, 1:3000 and 1:2000, respectively, Santa Cruz) and visualized using enhanced chemiluminescence.
8
Western Blot Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After the total proteins of cells in each group were isolated with total protein extraction kit (Transgen, DE101-01), protein samples were treated with SDS-PAGE protein loading buffer (Beyotime, P0015). Then the cell proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, which were transferred to a polyvinylidene fluoride membrane (Millipore), washed with TBST buffer (Solarbio, T1081). The membrane was sealed with 5% skim milk for 1h, and incubated with primary antibody at 4 °C overnight, and the second antibody incubated at 37 °C for 1h. Then, the samples were washed 3 times with TBST buffer (Solarbio, T1081), and ECL luminescence solution (Biosharp, BL520A) was added. The protein bands were observed using a chemiluminescence gel imaging system (Bio-RAD, USA). The used antibodies are as follows: GAPDH (Transgen, HC301-01), HSPA5 (Proteintect, 11587-1-AP), LC3B (Abcam, ab192890), P62 (BOSTER, M00300-1), Phospho-mTOR (Ser2448) (D9C2) (Cell Signaling Technology, 5536T), mTOR (7C10) (Cell Signaling Technology, 2983T), Phospho-Akt (Ser473) (D9E) (Cell Signaling Technology, 4060S), Akt (pan) (C67E7) (Cell Signaling Technology, 4691S), Exosome Panel (Calnexin, CD9, CD63, CD81, Hsp70, TSG101) (ab275018), Goat Anti-mouse IgG (Transgen, HS201-01), Goat anti-rabbit IgG (Transgen, HS101-01).
9
Antibody Characterization for Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following antibodies were used in our study. NELFB (Cell Signaling, 14894), NELFB (Proteintech, 16418-1-AP), NELFC (Cell Signaling, 12265), NELFE (Proteintech, 10705-1-AP), NELFA (Proteintech, 10456-1-AP), flag (Sigma, F3165), Tubulin (Millipore, 05-829), PI3 Kinase p110α (C73F8) (Cell Signaling, 4249), PI3 Kinase p110γ (D55D5) (Cell Signaling, 5405), Akt (pan) (C67E7) (Cell Signaling, 4691), p-Akt (Ser473) (D9E) XP (Cell Signaling, 4060), p-GSK-3β (Ser9) (D85E12) (Cell Signaling, 5558), GSK-3α/β (D75D3) (Cell Signaling, 5676), p-MEK1/2 (Ser217/221) (Cell Signaling, 9121), ERK1/2 (Cell Signaling, 4695), p-ERK1/2(Thr202/Tyr204) (Cell Signaling, 4370), ATP5B(Millipore, MABS1304), LaminB1(Santa Cruz, sc-374015), GAPDH (Bio-Rad, 12004167), ACTIN (Bio-Rad, 12004163), Vinculin (Proteintech, 66305), and Streptavidin-IRDye 800CW (Licor, 926-32230). Secondary antibodies include IRDye 680RD Goat anti-rabbit immunoglobulin G (Licor, 926-68071), IRDye 800CW Goat anti-mouse immunoglobulin G (Licor, 926-32210), and Goat anti-rabbit immunoglobulin G (H + L)-horseradish peroxidase (Invitrogen, 31460).
10
Immunodetection and Quantification of AKT and ER Stress Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies against phospho-Akt (Ser473) and Akt (pan) (C67E7) were obtained from Cell Signaling Technology (Danvers, MA). Recombinant human HSP70 and GRP78 were obtained from MyBiosource (San Diego, CA). Nile Red and 4',6-diamidino-2-phenylindole (DAPI) were obtained from Thermo Fisher Scientific (Vantaa, Finland). Plin2 antibody was obtained from LS-BIO (Seattle, WA), AlexaFluor 488 from Life Technology (Carlsbad, CA) and CD16-PeCy7 and CD14-ECD from Beckman Coulter (Brea CA). HSP70 (3A3) and GRP78 (76-E6) antibodies were obtained from Santa Cruz Biotechnology (Dallas, Texas) and Dynabeads® Protein A was obtained from Invitrogen (Waltham, MA, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!