goat anti human il2ra antibodies  (R&D Systems)

Journal: Biochimica et biophysica acta

Article Title: SAP97 blocks the RXR ER retention signal of NMDA receptor subunit GluN1-3 through its SH3 domain.

doi: 10.1016/j.bbamcr.2014.11.030

Figure Lengend Snippet: Fig. 1. Experimental strategy and background. (A) To assess the effect of PDZ proteins on GluN1-3 exit from the ER, the C-terminus was appended to VE (VE1-3). (B) The last two exons coding for the C-terminus GluN1 are alternatively spliced, and have either the C1 splice cassette, or not, joined to the C2 or C2′ splice cassette. The possible sequence determinants affecting ER retention and ER exit are shown for the different GluN1 C-termini. The PDZ-binding domains are underlined. The ER retention motif is indicated by three asterisks (***). PKC and PKA phosphorylation sites are shown as double-daggers (‡‡). GluN1-3 and GluN1-4 have a nested di-valine exit signal in the PDZ-binding motif, which has been shown to speed forward trafficking (++). (C) When cells are maintained at 40 °C, VE remains unfolded and chaperone-bound in the ER, but mobile [19], and when they are shifted to 32 °C, VE rapidly folds correctly and exits the ER in a wave, which can be assessed by: 1) changes in co-localization with subcellular markers, 2) Endo H sensitivity of two N-linked glycans on VE, and 3) antibody labeling to cell surface VE. Since the rate of ER exit and traversal of the secretory pathway can be accurately assessed, appending the GluN1-3 C-terminus to VE and co-transfecting with prospective interacting proteins were used to identify interacting proteins that could enhance ER-exit. (D) IL-2α has been used extensively as a trafficking reporter for analysis of trafficking determinants. An excellent monoclonal antibody hybridoma is available (7G7B6; ATCC) for immunofluorescence, ELISAs, and immunoprecipitation. For analysis of secretory trafficking by Western blot, IL2RA is extensively glycosylated such that the cell-surface form is about 25 kD larger than the immature form localized early in the secretory pathway.

Article Snippet: Goat anti-human IL2RA antibodies used for Western blots were from R&D Systems.

Techniques: Sequencing, Binding Assay, Phospho-proteomics, Antibody Labeling, Immunoprecipitation, Western Blot

Journal: Biochimica et biophysica acta

Article Title: SAP97 blocks the RXR ER retention signal of NMDA receptor subunit GluN1-3 through its SH3 domain.

doi: 10.1016/j.bbamcr.2014.11.030

Figure Lengend Snippet: Fig. 3. An SH3–GuK domain interaction is necessary for GluN1-3 ER exit. (A) Surface versus total ELISAs were performed at 3 h after switching cells to 32 °C with co-transfected VE1-3 and SAP97, or VE1-3 and cotransfected truncated segments of SAP97. Co-transfections of PDZ1-3 domains resulted in diminished surface expression. Successive removal of the GuK domain, and SH3–GuK domains of SAP97 resulted in a significant loss of cell-surface VE1-3 (**p b 0.05, ANOVA, post hoc comparisons), indicating that the SH3–GuK domains may be necessary to suppress ER retention of GluN1-3. (B) Expression levels of SAP97 and SAP97ΔSH3–GuK were assessed by immunoblotting transfections with anti-SAP97 antibody and anti-β-actin to control for load. No difference was observed. (C) Tac1-3Δ4 co-localized with GuK and SH3. Immunofluorescence micrographs of Tac1-3Δ4 co-expressed with EGFP control showed no shared edges and minimal co-localization (top row; scale bars: 10 μm). Tac1-3Δ4 co-expressed with EGFP–GuK showed striking co-localization (middle row). Tac1-3Δ4 showed co- localization with the SH3–GFP domain as well (bottom row; yellow arrows indicate shared edges). (D) Co-immunoprecipitation experiments (repeated 4 times with similar results) indicated that the SH3 and GuK domains interacted with the GluN1-3 C-terminus. Cells co-transfected with Tac1-3Δ4 and EGFP, EGFP–GuK, or SH3–EGFP were lysed, prepared, and subjected to immunoprecipitation with 7G7B6 antibody and SDS PAGE. Blots on the left were probed with anti-human IL2RA antibody to confirm immunoprecipitation. Blots on the right were probed with anti-GFP antibody. Panels show input (5%; In), I.P. (25%), and unbound (Un).

Article Snippet: Goat anti-human IL2RA antibodies used for Western blots were from R&D Systems.

Techniques: Transfection, Expressing, Western Blot, Control, Immunoprecipitation, SDS Page

Journal: Biochimica et biophysica acta

Article Title: SAP97 blocks the RXR ER retention signal of NMDA receptor subunit GluN1-3 through its SH3 domain.

doi: 10.1016/j.bbamcr.2014.11.030

Figure Lengend Snippet: Fig. 4. The SAP97 SH3 domain is sufficient to block ER retention. (A) Cell surface immunofluorescence immunolabeling of cells transfected with Tac1-3Δ4 and GFP (top row), or GFP–GuK (middle row), or SH3–GFP (bottom row) was performed as described in Materials and methods. Images were taken at low magnification (10×; scale bar: 100 μm) with the same settings for comparison. Left column shows GFP fluorescence, and the right column shows surface labeling of Tac1-3Δ4. Tac1-3Δ4 + GFP surface labeling showed background immunofluorescence (upper right picture), +GFP–GuK showed modest surface labeling coincident with GFP fluorescence (middle right; see arrows), and Tac1-3Δ4 showed saturating fluorescence on some cells that co-expressed SH3–GFP (bottom right; top two white arrows). This experiment was repeated more than 3 times with similar results. (B) Western blots of Tac1-3Δ4 alone or co-transfected with SH3–GFP, or GFP–GuK, full length SAP97, or full-length Tac1-3 probed with anti-IL2RA antibody (lanes indicated above blot) demonstrated that Tac1-3Δ4 alone showed only the immature, ER-localized band, whereas SH3–GFP co-expression caused a shift to mature Tac1-3Δ4 that was similar to the full length C-terminus, Tac1-3. Tac1-3Δ4 co-expression with full-length SAP97 showed only a small amount of mature form. (C) Surface versus total ELISAs with Tac1-3, and mutants, co-transfected with segments or chimeras of SAP97. Tac1-3 surface expression was significantly greater than Tac1-3Δ4 (††p b 0.01, one-way ANOVA, post hoc comparisons; N = number of independent transfections). Co-expression of Tac1-3Δ4 with the GFP–GuK domain and SH3–GuK–GFP domains resulted in no significant change in surface expression. Fusion of the N-terminal 120 amino acids with the SH3–GuK domain of SAP97 (Wu–GFP) co-expressed with Tac1-3Δ4 resulted in significant suppression of ER retention (†p b 0.05, ANOVA, post hoc comparisons). The SH3–GFP domain of SAP97 alone was sufficient to block ER retention of Tac1-3Δ4 (††p b 0.01, ANOVA, post hoc comparisons), confirming the results in (A) and (B). Co-expression of full-length SAP97 with Tac1-3Δ4 resulted in only a small and insignificant increase in surface expression. (D) Deletions, segments, and fusions of βSAP97 used in this study.

Article Snippet: Goat anti-human IL2RA antibodies used for Western blots were from R&D Systems.

Techniques: Blocking Assay, Immunolabeling, Transfection, Comparison, Labeling, Western Blot, Expressing