10–20 µg of total protein was separated with 7.5–12% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). The membranes were blocked with 5% non-fat milk (LabScientific, Highlands, New Jersey) in TBS for 30 min and then incubated with primary antibodies in TBS containing 5% milk and 0.1% Tween-20 overnight at 4°C. After rinsing with TBS-0.1% Tween, membranes were incubated with the secondary antibodies diluted in 5% milk-TBS for 1.5 hr at RT. Bands were detected by using Odyssey CLx Scanner. Band intensity quantifications were performed using Image Studio 3.1. Normalizations were performed either relative to the actin or Akt band intensities. Western blots were performed with antibodies against p110α (#4249), p110β (#3011), Akt (#4691), phospho-Akt T308 (#13038), phospho-Akt S473 (#9271), phospho-Erk 1/2 T202/Y204 (#9101), phospho-S6 S235/236 (#2211), phospho-S6 S240/244 (#5364), phospho-EGFR Y1068 (#2236), S6 (#2217), HA (#2367), TfnR (transferrin receptor) (#13208), Nup (nucleoporin) (#2598), EGFR (#4267), PTEN (#9188) (all Cell Signaling), Rac1 (05–389) (Millipore, Billerica, Massachusetts), β-actin (A5441), α-tubulin (T5168) (Sigma), Caveolin1 (MA3-600) (Thermo Fisher) and Gq (sc-392) (Santa Cruz). IRDye 800CW Goat anti-mouse or anti-rabbit secondary antibodies (Li-Cor, Lincoln, Nebraska) were used.
Irdye 800cw goat anti mouse or anti rabbit secondary antibodies
Manufactured by LI COR
Sourced in United States
The IRDye 800CW Goat anti-mouse or anti-rabbit secondary antibodies are fluorescently labeled reagents used for protein detection and quantification in Western blotting, immunohistochemistry, and other applications. The antibodies are conjugated to the near-infrared fluorescent dye IRDye 800CW, which provides high sensitivity and low background signal.
Automatically generated - may contain errors
Lab products found in correlation
Rac1 05 389, by Merck Group (1 mentions)
α tubulin t5168, by Merck Group (1 mentions)
β actin a5441, by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Mannitol, by Merck Group (1 mentions)
Histamine elisa kit, by Enzo Life Sciences (1 mentions)
Sp600125, by Cell Signaling Technology (1 mentions)
Histamine, by Merck Group (1 mentions)
β actin antibody, by Santa Cruz Biotechnology (1 mentions)
U0126, by Cell Signaling Technology (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
Sb203580, by Cell Signaling Technology (1 mentions)
D glucose, by Merck Group (1 mentions)
2 protocols using irdye 800cw goat anti mouse or anti rabbit secondary antibodies
1
Western Blot Analysis of PI3K/Akt Signaling
2
Histamine Signaling Pathway Regulation
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Mannitol, D-glucose and histamine were purchased from Sigma (St. Louis, Missouri, USA). The extracellular signal-regulated kinase (ERK) inhibitor, U0126, the c-Jun N-terminal kinase (JNK) inhibitor, SP600125 and the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor, SB203580 were obtained from Cell Signaling Technology (Danvers, Massachusetts, USA). Antibodies to the histamine receptor H4 (HRH4) were purchased from Abcam (Cambridge, Massachusetts, USA). Antibodies against p-ERK, ERK, p-JNK, JNK, p-p38 and p38 were obtained from Cell Signaling Technology. β-Actin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA). IRDYE 800CW goat antimouse or antirabbit secondary antibodies were from LI-COR (Lincoln, Nebraska, USA). VEGF and IL-6 ELISA kits were obtained from R&D Systems (Minneapolis, Minnesota, USA). The histamine ELISA kit was purchased from Enzo Life Sciences (Lausen, Switzerland). The histidine decarboxylase (HDC) ELISA kit was obtained from MyBioSource (San Diego, California, USA). All chemicals and buffers used in the study were purchased from Sigma.
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