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. Author manuscript; available in PMC: 2009 Jun 1.
Published in final edited form as: Mol Cell Neurosci. 2008 Mar 13;38(2):277–289. doi: 10.1016/j.mcn.2008.03.002

Figure 2.

Figure 2

Interaction between the γ2 subunit and CAML requires the cytoplasmic and 4th transmembrane domains of the γ2 subunit and the N-terminal cytoplasmic domain of CAML. A, B, Co-immunoprecipitation of CAML and GABAARs from lysates of transfected 293T cells. HEK 293T cells were cotransfected with FL-CAML and GABAARs (α2β3mycγ2) and cell lysates subjected to immunoprecipitation with anti-FLAG (A) or anti-myc antisera (B) or IgG as a negative control as indicated. Coimmunoprecipitation of GABAARs with anti-FLAG was dependent on cotransfected FL-CAML, as revealed by analysis of immunopurified samples with guinea pig anti γ2 antiserum (A). Conversely, copurification of FL-CAML with anti-myc mAb required cotransfection of GABAARs containing the mycγ2 subunit, as reveled by analysis of immunoblots with rabbit anti-CAML antibody (B). C, Endogenous CAML was immunoprecipitated from DSP-cross-linked mouse brain lysates with mAb anti-CAML and analyzed by western blot for coimmunoprecipitation of GABAARs using a rabbit anti α1 subunit antiserum (Benke et al., 1996). No coimmunoprecipitation was observed in reactions using unspecific IgG instead of anti-FLAG, anti-myc or anti-CAML (A-C). D, E, Interaction of CAML with the γ2 subunit required both cytoplasmic and 4th transmembrane domains of the γ2 subunit and the N-terminal cytoplasmic region of CAML. Various fragments derived from the C-terminal portion of the γ2 subunit (D) or α1 and β2 subunits of GABAARs (E) were tested as bait constructs for interaction with full-length CAML in yeast two-hybrid interaction assays. In parallel the γ2 subunit bait constructs were tested for proper interaction and with GABARAP(36-117) as a positive control. Construct names with numbers in parentheses refer to amino acid numbers of the mature subunit present in the construct. F, Full-length CAML and CAML(1-189) were analogously tested as a prey constructs for interaction with baits containing γ1(398-465), γ2(361-428) and γ3(400-467). Transmembrane regions of the γ2 subunit and CAML are indicated as black boxes. The region between M3 and M4 of the γ2 subunit and CAML(1-189) represent the cytoplasmic regions of the γ2 subunit and CAML, respectively. nd, not determined.