Western blotting of cell membrane protein preparations from first-, second-, and third-trimester placentas was performed to detect G-CSFR and actin (Fig. 4). Two distinct bands of 150 kDa and 120 kDa, which correspond to G-CSFR, could be detected with mAb LM832 in all first-and third-trimester placental proteins examined (Fig. 4a). These bands could also be detected in early second-trimester placental protein (13-13.5 wk); however, between 14 and 20 wk gestation, G-CSFR levels were low or undetectable (even after 30-min exposure of the filters to autoradiographic paper). There was a remarkably sharp drop in levels of G-CSFR protein between 13 and 14 wk gestation, which was particularly prominent in the samples analyzed in Figure 4a. One band of 43 kDa, which corresponds to actin, could be detected in all placental protein. The intensity of G-CSFR and actin bands was estimated by densitometry. The levels of G-CSFR protein were expressed as the ratio of the sum of the two G-CSFR band intensities to the actin band intensity as shown in Figure 4b. Levels of G-CSFR protein were 5-fold higher in third-trimester placental samples relative to first- and early second-trimester placentas (Fig. 4c; p = 0.0007). buy yasmin online
The levels of the two G-CSFR immunoreactive bands were estimated by densitometry and expressed as the ratio of the 150-kDa band to the 120-kDa band (Fig. 4d). The values obtained when the ratios of the two bands were compared were consistently higher in the third trimester, with a 5-fold increase in intensity relative to first-trimester levels (p = 0.0007).
FIG. 4. Semiquantitative Western blotting of G-CSFR and actin in first-, second-, and third-trimester placental membrane protein extracts. a) Western blot of tissue extracts from placentas of different gestational age (weeks). LM832 recognized two high-molecular mass bands of —120- and 150-kDa in samples from Weeks 6-13.5 and 29-40, but not from Weeks 14 to 20 gestation. Actin (43 kDa) was detected in all samples. b) Densitometric analysis of G-CSFR protein levels in placental tissue samples assayed in a. Levels of G-CSFR protein were determined as described in the text. c) Bar graph representing the mean G-CSFR protein values obtained from densitometric analysis from b, ± SEM. d) Densitometric analyses of the two bands corresponding to G-CSFR in the gels shown in a. The intensities of bands corresponding to G-CSFR were expressed as a ratio of the 150-kDa band to the 120-kDa band. The data presented are representative of three independent experiments.