Total RNA isolated from neurons and MEF was analyzed using RT-qPCR for the presence of IFNγR1 and IFNγR2 as described in Materials and Methods.

Neurons and MEF were treated as described in Figure 5a above. Total RNA was purified from the lysates and analyzed using RT-qPCR for the presence of CXCL10 (a), IRF-1 (b), SOCS-1 (c), and SOCS-3 (d) transcripts. The timepoints correspond to the length of time in h after the IFNγ was washed out.

a) Quantitative timecourse immunoblot analysis of purified total protein (20 μg/lane) isolated from untreated and IFNγ-treated (100 U/ml) neurons and MEF using antibodies specific for phospho-STAT3, STAT3, and GAPDH. Blots were first analyzed for phospho-STAT3 and GAPDH, and were then stripped and analyzed for STAT3. b) Neurons and MEF were prepared as described, but treated with 250 ng/ml IL-6. Lysates were collected at the indicated timepoints, and were subsequently immunoblotted with antibodies to phospho-STAT3 and GAPDH.

Total RNA isolated from neurons and MEF treated with IFNγ (100U/ml) for the indicated times was analyzed using RT-qPCR for the presence of CXCL10 (a), IRF-1 (b), SOCS-1 (c), and SOCS-3 (d) transcripts. The top plot for each gene shows transcript levels from untreated cells and cells treated for 0.5 – 6 h; the bottom plot shows the entire time course.

a) Neurons and MEF were treated with IFNγ (100U/ml) for 30 min, then washed extensively to eliminate IFNγ from the cultures. Conditioned culture medium was replaced after washing, and cells were lysed at the indicated timepoints post-treatment. b) Equal volumes of whole cell lysates were examined by immunoblotting with anti-phospho-STAT1 and anti-GAPDH antibodies. The timepoints correspond to the length of time in h after the IFNγ was washed out. Shown are results from a simultaneous exposure of the blots.

The kinetics of STAT1 phosphorylation in response to IFNγ (100U/ml) were examined in neurons and MEF. a) Quantitative timecourse immunoblot analysis of i) purified total protein (20 μg/lane) isolated from untreated and IFNγ-treated neurons and MEF, and ii) samples from whole cell lysates of equal numbers (5.32×10⁵ cells per condition) of neurons and MEF, using antibodies specific for phospho-STAT1 (pY701), STAT1, and GAPDH. Blots were first analyzed for phospho-STAT1 (pY701) and GAPDH, and were then stripped and analyzed for STAT1. The presence of phospho-STAT1 in the apparent absence of total STAT1 in neurons is likely due to differences in antibody affinity. b) “Per-molecule” response of STAT1 activation in neurons and MEF as assessed by normalization of phospho-STAT1 signal to STAT1 and GAPDH signals. Data from two timecourse studies similar to that described in (a) above was analyzed using densitometry; mean normalized phospho-STAT1 signal is shown. c) Densitometric quantitation of 48 h IFNγ timecourse. Purified total protein (20 μg/lane) isolated from untreated and IFNγ-treated neurons and MEF was analyzed by immunoblot with antibodies specific for phospho-STAT1 and GAPDH. The phospho-STAT1 signal was normalized to the GAPDH signal to control for loading, and is presented as percent maximum phospho-STAT1 signal. d) Densitometric quantitation of STAT1 phosphorylation in response to continual IFNγ stimulation in neurons 1, 5, and 8 d post-plating, and in MEF. IFNγ was added to cultures, whole cell lysates were collected at indicated timepoints post-IFNγ addition, and the lysates were analyzed by immunoblot with antibodies specific for phospho-STAT1 and GAPDH. The phospho-STAT1 signal is presented as described in (a).