rabbit polyclonal antibody against mouse eea1  (Cell Signaling Technology Inc)

Journal: Microorganisms

Article Title: Early Intracellular Trafficking and Subsequent Activity of Programmed Cell Death in Channel Catfish Macrophages Infected with Edwardsiella ictaluri

doi: 10.3390/microorganisms8111649

Figure Lengend Snippet: Immunofluorescence micrographs showing subcellular co-localization of early and late endosomal markers. WT and 65ST strains of E. ictaluri stain green. The host cell nucleus stains blue. The host cell is visible in DIC in the merged image. The graphs show the percentage of the ECV that col-localized with the given marker over time. ( A ) The early endosomal markers Rab 5 and EEA1 stain red. ( B ) The late endosomal markers Rab7 and Lamp1 also stain red. Results show that %-co-localization of the T3SS mutant 65ST and WT were not significantly different for the early endosomal markers Rab5 and EEA1, or for the late endosomal marker Rab7. Significantly lower levels of Lamp1 in the WT, however, indicate that phagosomal/lysosomal fusion did not occur for the WT. Statistical significances over time were determined using one-tailed unpaired T-test for each period. Data presented as the mean of three sets of 100 cells. Asterisks indicate significant differences of the WT compared to the TT3S mutant 65 ST (* = p ≤ 0.01, *** = p ≤ 0.001).

Article Snippet: Rabbit polyclonal antibody against early endosomal antigen 1, EEA1, the small GTPase, Rab5, and the endoplasmic reticulum marker, calnexin, were purchased from GenScript (Piscataway, NJ, USA, Catalogue # A01514, A01302, and A01192, respectively).

Techniques: Immunofluorescence, Staining, Marker, Mutagenesis, One-tailed Test

Journal: Nature Communications

Article Title: Vps11 and Vps18 of Vps-C membrane traffic complexes are E3 ubiquitin ligases and fine-tune signalling

doi: 10.1038/s41467-019-09800-y

Figure Lengend Snippet: Regulation of ERα activity by Vps11/18 is independent of intracellular trafficking pathways. a ERα reporter gene assays with HEK293T cells overexpressing Vps11/18 in combination with wild-type (Wt) or K44A mutant (Mut) dynamin II (mean ± s.e.m. with n = 3 biologically independent experiments). b Assays as in a but with knock-downs with the indicated shRNA constructs (mean ± s.e.m. with n = 3 biologically independent experiments). c Transferrin uptake assays in HEK293T cells overexpressing Vps11/18 along with cyan fluorescent protein (CFP). Scale bar indicates 20 µm. d Transferrin uptake assays with immunostaining of EEA1 or LAMP1 in HEK293T cells overexpressing Vps11/18 and blue fluorescent protein (BFP). Scale bar indicates 20 µm. e Assays as in a with knock-downs of HRS or TSG101 (mean ± s.e.m. with n = 3 biologically independent experiments). f – i Assays as in a under the following conditions: treatments to inhibit (Wortmannin, 3-methyladenine (3-MA)) or to stimulate (serum starvation, rapamycin) autophagy ( f and g , respectively); knock-down of SQSTM1 ( h ); treatment with the EGFR inhibitor AG1478 ( i ) (mean ± s.e.m. with n ≥ 3 biologically independent experiments). j , k Immunoblots displaying PKA substrates and protein levels with or without FI ( j ) or phospho-ERK1/2 and ERK1/2 protein levels with or without FBS ( k ) in HEK293T cells overexpressing Vps11/18; FI, cocktail of forskolin and isobutylmethylxanthine to increase intracellular cAMP levels; FBS, fetal bovine serum. l Assays as in a with overexpression of the transcriptional coactivators SRC1 or CARM1 (mean ± s.e.m. with n = 3 biologically independent experiments). Asterisks indicate significant differences with the corresponding negative controls with p -values < 0.05. Statistical significance was determined with unpaired and two-sided Student’s t -tests. Source data are provided as a Source Data file

Article Snippet: The rabbit polyclonal antisera against ERα (HC-20, sc-543, discontinued) and p-ERα (Ser118, sc-12915-R, discontinued), the mouse monoclonal antibodies against ERK1/2 (C-9, sc-514302), p-ERK (E-4, sc-7383), Vps11 (S-38, sc-100893) and SUMO-2/3/4 (C-3, sc-393144), and the goat polyclonal anti-Vps16 (C-17, sc-86939, discontinued) were from Santa Cruz Biotechnologies (all diluted 1/200 for immunoblots); the rabbit polyclonal antisera against PELP1/MNAR (A300-180A) and BCAR/p130Cas (A301-667A) were from Bethyl Laboratories (all diluted 1/500 for immunoblots); the mouse monoclonal anti-GAPDH (6C5, ab8245) was from Abcam (diluted 1/30,000 for immunoblots); the mouse monoclonal anti-HA.11 (16B12, MMS-101P) was from Biolegend (for immunoprecipitations, 2 µg of antibody was used for 2 mg of proteins); the rabbit polyclonal antisera against Vps33A (PA545268), p-ERα (Ser167, PA537570) and p-ERα (Tyr537, PA537571), the mouse monoclonal antibody against Vps18 (4E9, MA522391) were from Thermo Fisher Scientific (all diluted 1/500 for immunoblots; for immunoprecipitations, 2 µg of anti-Vps18 antibody were used for 2 mg of proteins); the rabbit polyclonal antiserum against PKA substrates (P-(S/T), 9621), the rabbit monoclonal antibody against phospho-Src (Tyr416) (D49G4, 6943) (both diluted 1/500 for immunoblots), and the PTMScan Ubiquitin Remnant Motif (K-ε-GG) reagents were from Cell Signaling Technology; the rabbit polyclonal antiserum against PKA (06–903) was from Upstate (diluted 1/500 for immunoblots); the mouse monoclonal antibody against c-Src (GD11, 05–184) was from Millipore (diluted 1/500 for immunoblots); the rabbit polyclonal antiserum against SUMO-1 was from Alexis Biochemicals (BML-PW0505A, diluted 1/500 for immunoblots); the rabbit polyclonal antibody against human EEA1 (ALX-210–239) was from Enzo (diluted 1/100 for immunofluorescence), and the mouse monoclonal antibody against human LAMP1 (H4A3) was from BD PharMingen (diluted 1/100 for immunofluorescence).17β-estradiol (used at 100 nM), dexamethasone (used at 100 nM), progesterone (used at 100 nM), phorbol myristate acetate (PMA) (used at 1 µg/ml), cobalt (II) chloride (used at 100 µM), wortmannin (used at 1 µM), 3-methyladenine (used at 5 mM), rapamycin (used at 1 µM), forskolin (used at 10 µM), isobutylmethylxanthine (used at 100 µM), chloroquine (used at 50 µM), brefeldin A (used at 5 µg/ml) and AG1478 (used at 10 µM) were from Sigma-Aldrich; MG132 (used at 5 µM) was from Enzo Life Sciences.

Techniques: Activity Assay, Mutagenesis, shRNA, Construct, Immunostaining, Western Blot, Over Expression