Bovine serum albumin (BSA), Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), penicillin, RPMI-1640, streptomycin, and trypsin were purchased from Gibco-BRL (Grand Island, NY, USA). Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT), and propidium iodide (PI) were from Sigma-Aldrich Inc. (St. Louis, MO, USA). Antibodies for caspase-7, caspase-9, caspase-3, Bcl-xL Mcl-1, PARP, Bcl-2, p-Akt, p-PI3K, p-PDK, p-PTEN, p-mTOR, p-p70S6K, p-S6K, p-4EBP1, p-eIF4E, and GAPDH were obtained from Cell Signaling Technology (Beverly, MA, USA). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) System Kit was procured from Calbiochem (Darmstadt, Germany). ALT, AST, and CK were obtained from IDEXX Laboratories Inc. (Westbrook, Maine USA). All other reagents used were of the purest grade available.
P pdk
Manufactured by Cell Signaling Technology
Sourced in United States
P-PDK is a lab equipment product offered by Cell Signaling Technology. It functions as a phosphorylation-specific antibody that detects the phosphorylated form of PDK1 (phosphoinositide-dependent kinase-1).
Automatically generated - may contain errors
Lab products found in correlation
P akt, by Cell Signaling Technology (3 mentions)
P mtor, by Cell Signaling Technology (2 mentions)
P pi3k, by Cell Signaling Technology (2 mentions)
Gapdh, by Proteintech (2 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
P s6k, by Cell Signaling Technology (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt dimethyl sulfoxide dmso, by Merck Group (1 mentions)
P p70s6k, by Cell Signaling Technology (1 mentions)
P pten, by Cell Signaling Technology (1 mentions)
P eif4e, by Cell Signaling Technology (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
P 4ebp1, by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Cd133, by Proteintech (1 mentions)
P c raf, by Cell Signaling Technology (1 mentions)
Nanog, by Proteintech (1 mentions)
Cyclooxygenase 2 cox2, by Cell Signaling Technology (1 mentions)
6 protocols using p pdk
1
Cytotoxicity Evaluation and Apoptosis Pathway
2
Investigating 5-FU and ELE Mechanisms
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5-FU was obtained from Sigma Aldrich (St. Louis, MO, USA), and ELE (99.2% purity) was acquired from the Chinese National Institute for Drugs and Food Control (Liaoning, China). The primary antibodies of vimentin, N-cadherin, E-cadherin, mitochondrial membrane potential (MMP)-9, cyclooxygenase 2 (COX-2), p-110α, p-110β, AKT, p- AKT (Ser473), p-PDK (Ser421), p-cRaf (Ser 259), ERK, p-ERK, p38, p-P38, IkB-α, p-IkB-α, p-IKKα/β, IKKα, p-50, p65, and p-P65 were acquired from Cell Signaling Technology, Inc. (USA). Cleaved (cl) PARP, cl-caspase-3, Bcl-2, Bax, CD133, Nanog, CD44, OCT4, β-actin, and GAPDH were purchased from Protein tech (Wuhan, China). Other chemicals were ordered from Sigma unless specified otherwise.
3
Immunohistochemical Analysis of Breast Cancer
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Tissue microarray of breast cancer, Paraffin sections and mouse xenograft tumors were deparaffinized and rehydrated followed by antigen retrieval in citrate buffer. After blocking with serum, FUT4 antibody (1:100), p-PDK (1:50), p-Akt (1:50) (Cell Signaling Technology, Danvers, USA) were incubated overnight at 4 °C. Then the tissue slides were incubated with anti-rabbit horseradish peroxidase-conjugated antibody for 45 min. The slides were stained and visualized by using 3,3′diaminobenzidine solution. Images were taken with microscope. The tissue section staining was analyzed blindly by at least two pathologists and cell scores were obtained. The expression level was divided into two categories: weak (cell score below average) and high (cell score above average).
4
Western Blot Analysis of Signaling Proteins
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Antibodies were purchased for the detection of β-actin (AC-15; Sigma); PRAS40 (Invitrogen); cleaved PARP, p-Akt (S473), p-PDK1, p-PRAS40 (T246), p-PI3K (p85), p-S6 ribosomal protein, p-mTOR (S2448), S6 ribosomal protein, p-IGF1R (Y1135/1136)/p-IR (Y1150/1151), S6, Akt, PI3K (p85), mTOR, IGF1R and IR (Cell Signaling); p-IR (Y1361, Abcam); p-ERK1/2 and ERK1/2 (Abbkine). Western blot analysis was performed as described previously (Huang et al. 2014 (link)), and the signals were detected using an ECL Plus Detection System (Thermo Fisher). Images were acquired using an Image Analyzer ChemiDoc XRS+ (Bio-Rad) and quantified with Image J software.
5
Whole-Cell Protein Lysate Preparation
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To prepare whole‐cell protein lysates, cells at 90% confluence were washed in PBS before incubation with RIPA lysis buffer. Equal protein was loaded onto 10% SDS‐PAGE gels, transferred onto nitrocellulose membranes and blocked with TTBS containing 5% fat‐free dry milk. The membranes were incubated at 4°C overnight with the primary antibodies: uPA and uPAR (Abcam), p‐c‐FOS (Ser32), p‐c‐JUN (Ser73), c‐FOS, c‐JUN, Akt, p‐Akt (Tyr308), PDK and p‐PDK (Ser241) (Cell Signaling Technology) and CK‐7, vimentin, N‐cadherin, E‐cadherin, poFUT1, HLA‐G and GAPDH (Proteintech). Next, the membranes were incubated with HRP‐conjugated goat anti‐rabbit IgG, HRP‐conjugated goat anti‐mouse IgG or HRP‐conjugated streptavidin for 1 hour. An enhanced chemiluminescence detection system (Bio‐Rad) was used to visualize immunoreactive bands.
6
Western Blot Analysis of Hippocampal Proteins
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Hippocampal tissue was homogenized on ice and lysed in a radioimmunoprecipitation (RIPA) cell lysis buffer (1X) with ethylenediaminetetraacetic acid (EDTA). Protein content was measured using a Bio-Rad colorimetric protein assay kit (Bio-Rad, Hercules). We separated 20 µg of protein on SDS-polyacrylamide gels and transferred the protein onto a nitrocellulose blotting membrane, which was incubated with the following primary antibodies: mouse β-actin (1:1000; Santa Cruz Biotech), rabbit BDNF (Santa Cruz Biotech, sc-20981), TrkB (1:1000; Santa Cruz Biotech, sc-119), IR (1:1000; cell signaling, #3095), p-IR (Tyr1162/1163) (1:1000; cell application, CB 486), PI3K (Santa Cruz Biotech, sc-1637), p-PI3K (Tyr467, sc-293115) (Santa Cruz Biotech, sc-293115) PDK (cell signaling, #3062), p-PDK (Ser241) (1:1000; cell signaling, #3061), AKT (Santa Cruz Biotech, sc-8312), p-AKT (Ser 473) (Santa Cruz Biotech, sc-135651), GSK-3β (cell signaling, #9315), and p-GSK3β (Ser 9) (1:1000; cell signaling, #9323). Horseradish peroxidase-conjugated anti-rabbit secondary antibody was used for BDNF, TrkB, IR, p-IR, PDK, p-PI3K, p-PDK, AKT, p-AKT, GSK-3β, and p-GSK-3β and anti-mouse secondary antibody was used for PI3K and β-actin. Experiments were performed under normal lab conditions and at room temperature (except for the transferred membrane).
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