Regulators and Effectors of Arf GTPases in Neutrophils

Figure 5

Expression of AGAP2 protein in PMNs. (a) Cells were lysed in nondenaturing isotonic (1% NP-40) or hypotonic (0.1% NP-40) lysis buffer. (b) PMNs were lysed in nondenaturing isotonic lysis buffer and AGAP2 was immunoprecipitated with preimmune serum or immune serum raised against AGAP2. Cell lysates derived from 1.5 × 10⁶ PMNs (a) and immunoprecipitates (b) were resolved by SDS-PAGE. Purified His6-tagged AGAP2 was used as a control. (c) Amino acid sequence of human AGAP2 (NCBI Reference Sequence: NP_055585.1). AGAP2 was immunoprecipitated from PMNs (4 × 10⁷/mL) as described in (b). The samples were resolved using 7.5–20% SDS-PAGE and the gel was stained with SYPRO Ruby. Bands of interest were analysed by mass spectrometry. The peptides identified by mass spectrometry are in red or blue. Peptides unique to AGAP2 are underlined.

(a)

(b)

(c)