The following antibodies were used: Anti-CXCR7 (GTX100027) (Genetex); Anti-EGFR (A300–387) from Bethyl; Anti-EEA1 (610456) from BD Biosciences; Anti-HDAC1 (ab11966), Anti-H3 (ab1791) and anti-GAPDH (ab9385) from Abcam; Anti-p53 (SC126) and anti-α-tubulin (DM1A) from Santa Cruz; Monoclonal anti-Flag (M2) (F1804) and polyclonal anti-Flag (F7425) from Sigma; Anti-Ki-67 (9027),anti-Phospho-MEK1/2 (Ser217/221) (9154), anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (4370), anti-p44/42 MAPK (Erk1/2) (4695), anti-PSA (2475), and anti-ARRB2 (3857) are purchased from Cell Signaling. Anti-CXCR7 (MAB42273) and anti-CXCR4 (MAB170SP) are purchased from R&D systems. pHAGE-Flag-CXCR7 construct was made through subcloning CXCR7 ORF into pHAGE-Flag vector. ARRB2 was first cloned into gateway entry vector pCR8, then transferred into destination vector pLentiSFB with LR clonase (Invitrogen). CXCR7p and CXCR7e guide RNA were subcloned into Lenti-CRISPRV2 vector, CXCR7 shRNAs (TRCN0000014509, TRCN0000363205), ARRB2 (TRCN0000165387, TRCN0000280686) and CXCR4 shRNA (TRCN0000256866) in pLKO vector were purchased from Sigma. Packed lentiviral particles were collected from 293T cultures 48 hours post transfection with lentiviral constructs, pSPAX2 and pMD2G. Viral supernatants were passed through a 0.45-mmfilter, supplemented with 4 ug/mL polybrene, and used to infect prostate cancer cells.
Anti eea1 610456
Manufactured by BD
Anti-EEA1 (610456) is a primary antibody used in laboratory research. It specifically binds to the Early Endosome Antigen 1 (EEA1) protein, which is a marker for early endosomes in cells. This antibody can be used in various applications, such as immunofluorescence, immunohistochemistry, and Western blotting, to detect and study the localization and expression of EEA1 in biological samples.
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2 protocols using anti eea1 610456
1
Antibody Characterization for Prostate Cancer
2
Immunohistochemical Staining of Subcellular Organelles
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Cells were fixed with 4% formaldehyde for 15 min at room temperature, washed three times with PBS and permeabilized in PBS containing 0.2% BSA and 0.05% saponin. Cells were then incubated with the primary antibodies in PBS containing 0.2% BSA and 0.05% saponin (anti-Lamp1/CD107a, 555798, BP PharmingenTM, 1:1000; anti-mTOR, 7C10, #2983, Cell Signaling Technology, 1:1000; anti-EEA1, 610456, BD Biosciences, 1:500; protrudin/ ZFYVE27, 12680-1-AP, Proteintech, 1:500) and Alexa Fluor 488, Alexa Fluor 647 or Cy3-coupled secondary antibodies (Jackson ImmunoResearch, 1:400 in PBS containing 0.2% BSA and 0.05% saponin) for 1 h. Actin was visualized by FluoProbes 547H (557/572 nm) coupled Phalloïdin (Interchim) and nuclei with 0.2 μg/mL 4’,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Coverslips were mounted in Mowiol (Sigma-Aldrich).
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