200 nM purified GSK3β was incubated with 12 μM tyrosine phosphatase (YopH) for 0.5, 1, or 4 hrs in PMP buffer (New England Biolabs) [50 mM HEPES pH 7.5, 100 mM NaCl, 2 mM DTT, 0.01% Brij 35] at 25 °C (Figure S1B ). The level of Tyr216 phosphorylation was assessed by western blotting using a primary Anti-GSK-3β (pY216) antibody (BD Biosciences #612312) and a secondary IRDye 800CW Donkey Anti-Mouse IgG antibody (Li-Cor #926-32212). Total GSK3β (MBP-tagged) was detected by western blotting using a primary MBP Tag (8G1) antibody (Cell Signaling Technology #2396) and a secondary IRDye 800CW Donkey Anti-Mouse IgG antibody (Li-Cor #926-32212).
Irdye 800cw donkey anti mouse igg antibody
Manufactured by LI COR
Sourced in United States
The IRDye 800CW Donkey Anti-Mouse IgG antibody is a secondary antibody labeled with the near-infrared dye IRDye 800CW. It is designed to detect and bind to mouse immunoglobulin G (IgG) antibodies in various immunoassays and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Pmp buffer, by New England Biolabs (1 mentions)
Anti mbp antibody, by Cell Signaling Technology (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Calyculin a, by Merck Group (1 mentions)
Phos tag acrylamide aal 107, by Fujifilm (1 mentions)
Rabbit anti β actin antibody, by Cell Signaling Technology (1 mentions)
Odyssey blocking buffer tbs, by LI COR (1 mentions)
Irdye 680lt goat anti rabbit igg antibody, by LI COR (1 mentions)
Immulon 4hbx, by Thermo Fisher Scientific (1 mentions)
Odyssey imager, by LI COR (1 mentions)
4 protocols using irdye 800cw donkey anti mouse igg antibody
1
Phosphorylation of GSK3β by Tyrosine Phosphatase
2
Phosphate-Affinity Gel Electrophoresis
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Phos-tag gels were prepared and run as previously described (Gavagan et al., 2020 (link)). After electrophoresis, the gel was incubated 3 x with transfer buffer +10 mM EDTA for 10 min before the transfer to increase transfer efficiency. Protein levels were detected with anti-MBP antibody (Cell Signaling Technology #2396; Figure 2—figure supplement 9A ). The secondary antibody was IRDye 800CW Donkey Anti-Mouse IgG antibody (Li-Cor #926–32212).
3
Phosphoprotein Analysis of Cellular Signaling Pathways
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Calyculin A (no. C5552) was purchased from Sigma-Aldrich (St. Louis, MO). Okadaic acid was from Calbiochem (no. 459620). Phos-tag acrylamide AAL-107 (no. 304–93521) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The following antibodies were purchased from Cell Signaling Technology (Danvers, MA): Anti-p44/42 MAPK (Erk1/2) rabbit antibody (no. 4695), anti-phospho-p44/42 MAPK (Erk1/2) mouse antibody (no. 9106S), anti-S6 ribosomal protein rabbit antibody (no. 2317S), anti-phospho-S6 ribosomal protein (Ser235/236) rabbit antibody (no. 4858S), anti-phospho-Akt (Ser473) mouse antibody (no. 4060), anti-phospho-Akt (Thr308) rabbit antibody (no. 13038S), anti-pan Akt rabbit antibody (no. 4691S) and anti-β-actin rabbit antibody (no. 4790S). Anti-α-tubulin mouse antibody was from Calbiochem (no. CP06). Odyssey Blocking Buffer (TBS) (no. 927–50000; LI-COR Biosciences), IRDye680LT goat anti-rabbit IgG antibody (no. 925–68021) and IRDye800CW donkey anti-mouse IgG antibody (no. 925–32212) were obtained from LI-COR Bioscience (Lincoln, NE).
4
Quantifying Antigen-Specific Antibodies via ELISA
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The amount of serum antibodies specific to the target antigen were assessed by either ELISA (OVA) or cell-based ELISA (HER2 and HER3) as previously described.22 27 (link) Briefly, ELISA for anti-OVA antibodies in serum was performed using OVA protein (2.5 µg/50 µL/well) coated plates (Immulon 4HBX, Thermo Scientific, Massachusetts, USA). Cell-based ELISA was performed using 96-well plates seeded with BC cells (4T1-HER2, 4T1-HER3, or parental 4T1, 10,000 cells/well). IRDye 800CW Donkey anti-mouse IgG antibody (1:2000 in 1%BSA-PBS, LI-COR Biosciences, Lincoln, Nebraska, USA) was added and plates were analyzed using LI-COR ODYSSEY imager (LI-COR). Detailed methods are shown in online supplemental method .
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