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. 2018 Dec 17;6(4):53. doi: 10.3390/proteomes6040053

Figure 1.

Figure 1

Generation and characterization of Cre-expressing, HA-tagged human spinophilin mice. (A) A construct containing DNA encoding HA-tagged human spinophilin with a P2A sequence and mNeptune3 fluorescent protein was cloned into the pBIGT vector that contains a floxed-stop sequence. (B) The construct encoding the floxed-stop sequence and the HA-spinophilin-P2A-mNeptune 3 sequence was subcloned into the ROSA targeting vector pROSA_26.PA. (C) The modified ROSA vector was used for generation of the targeted transgenic mice. (D) Striatal cells were transfected without or with HA-tagged human spinophilin. Lysates were immunoprecipitated with either an HA or spinophilin antibody and immunoblotted with an HA antibody or a spinophilin antibody. HA-spinophilin was selectively detected when it was overexpressed. (E) Mice express HA-tagged spinophilin upon crossing with Cre recombinase expressed in the direct pathway (D1) or indirect pathway (A2A) medium spiny neurons. (F) Spinophilin and protein phosphatase 1 immunoblots of inputs and HA-immunoprecipitates from HA spinophilin mice crossed with D1 or A2A Cre-recombinase-expressing mice. (G) Mice expressing HA-spinophilin had non-significant increases in total spinophilin expression.