Supplementary Figure 5: SCRIB regulated inflammatory response.

(a) Bone marrow cells were isolated from ishSCRIB mice with normal food. Cells were split into two groups: in present of Doxycycline (1 μg ml⁻¹) or not in present of Doxycycline. After 7 day of culture, cells were treated with LPS (100 ng ml⁻¹) for 2.0 h. RNA were isolated from untreated cells and treated cells and then followed by reverse transcripts PCRs. cDNA were used for Taqman array mouse inflammation gene set. Data shown is average of results from 2 mice. (b) Lysates from RAW 264.7shLuc (control) or shScrib treated with LPS (100 ng ml⁻¹) for indicated time point, immunoblotted for p-IkBα, IkBα, and Actin. (c) Confocal microscope images of RAW 264.7 cells before or after stimulated with LPS for 5 min, 15 min, 30 min or 3 h, fixed and stained for p65 (green) and DAPI (blue). Representative images were shown on the left panel (scale bars represent 50 μm) and quantification of p65 translocate to the nuclear were shown on the bottom right panel. n = 3 biologically independent experiments (mean ± SD). (two-tailed, unpaired t-test with equal variances: P = 0.01). BMDCs from WT or ishSCRIB mice were grown as described in Methods with or without Dox. On day 8, DCs were harvested and stimulated with LPS d or CpG e for 18h at the concentrations indicated. Data shown is representative of reproducible results from 2 mice d DC cells were then stained for indicated markers and analyzed by flow cytometry. Unprocessed scans of full blots/gels are shown in Supplementary Fig. 6.