FGFs are cleaved in S2 cell culture and embryonic extracts. (A) In vitro transcription/translation incorporating S35Met into Pyr (left) and Ths (right) supports production of ~80 kD proteins, as predicted from the sequence. (B) Schematics of HA-Pyr-Myc and HA-Ths-Myc constructs showing the position of the signal sequence (orange box), N-terminal HA-tag (blue box), FGF domain, and C-terminal Myc tag (green box). Upon transfection of S2 cells, HA-Pyr-Myc and HA-Ths-Myc are secreted from cells as multiple bands around 50 kD for Pyr (lane 2) and 35 kD for Ths (lane 3), as detected by immunoprecipitation and immunoblot with anti-HA to track N-termini. Lane 1 and 4 are supernatant and cell pellet controls transfected with empty vector (i.e., pUASt). Lane 5 and 6 are immunoprecipitations of HA-Pyr-Myc and HA-Ths-Myc from the cell pellet, showing cleaved forms are already detectable inside the cell. (C) Extracts from wildtype embryos (yw) or embryos overexpressing HA-Ths (Ths) or HA-Pyr (Pyr) with the 69B-GAL4 driver, immunoprecipitated with rat anti-HA and detected with mouse anti-HA reveal cleaved bands around 35 kD for Ths and around 45 kD for Pyr. (D) (Left Blot) Supernatant (i.e., cell culture medium) from HA-Pyr-Myc and HA-Ths-Myc, without immunoprecipitation, blotted with anti-HA antibody, shows a full-length band in the supernatant for Ths but not Pyr; the full-length Ths protein is present at much lower levels than the cleaved form and is only observable upon longer exposure; for instance, in (B), lane 3, it is not detected. (Right Blot) Immunoprecipitating with anti-Myc and blotting with anti-HA shows that the 150 kD band in Ths supernatant and cell pellet has both the N- and C-terminus connected.
Tulin and Stathopoulos BMC Developmental Biology 2010 10:83 doi:10.1186/1471-213X-10-83