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IKKβ and mutant huntingtin interactions regulate the expression of IL-34; implications for microglial-mediated neurodegeneration in HD
Ali Khoshnan1, Adam Sabbaugh1, Barbara Calamini2, Steven A. Marinero2, Denise E. Dunn2, Jung Hyun Yoo1, Jan Ko1, Donald C. Lo2, and Paul H. Patterson1
1Biology and Bioengineering, California Institute of Technology, Pasadena CA 91125
2Center for Drug Discovery and Department of Neurobiology, Duke University Medical Center, Durham, NC 27710
Figure S1. HTTx1-EGFP (25Qs and 103Qs) expressed in 10-days old MESC2.10
neurons. Anti-GFP antibody was used to enhance detection of the fusion proteins
(green). WT HTTx1 remains soluble where as HTTx1-103Q forms inclusion bodies as
the neurons age.
Figure S2. qRT-PCR showing levels of IL-34 mRNA in the brains of 3 months old R6/2
(A) and 12 months old YAC-128 (B) mice. All animal experiments and care complied
with federal regulations and were reviewed and approved by the Institutional Animal
Care and Use Committee and Animal Guidelines at the California Institute of
Technology. Briefly, striatal brain tissues were collected according to standard
procedures. RNA was extracted and examined by qRT-PCR using Taqman probes
(ThermoFisher). Data are shown as average fold-changes + SD, normalized to GAPDH
levels. (n=4 mice in each group). **p < 0.01.
Supplementary Figure 3
Figure S3. Western blot (WB) showing effective KD of IKKβ in MESC2.10 neuronal
progenitor cells. Engineering of NPCs with KD of IKKβ was reported previously (25).
Figure S4. WB analyses of lysates from neurons expressing mHTTx1 or mHTTx1 with
KD expression of IKKβ probed with two newly generated mouse polyclonal antibodies
against mHTTx1 (Parts A and B, respectively). Results confirm data in Fig. 3 that KD of
IKKβ blocks accumulation of high-molecular weight mHTTx1 aggregates in developing
neurons (right lanes in each blot). N.S.=non-specific.
Supplementary Figure 5
Figure S5.
Part A shows RT-qPCR quantification of mHTTx1 mRNA in proliferating and
differentiating NPCs expressing mHTTx1 and mHTTx1 with KD expression of IKKβ.
SYBR green was used to quantify the mRNA. Data were adjusted to values obtained for
actin. (B) Visualization of mHTTx1 RT-PCR products by agarose gel electrophoresis
and ethidium bromide staining. (C) WB showing that KD of IKKβ has no effect on the
levels of mHTTx1 monomers in proliferating NPCs (lanes 0, arrow) detected by an
antibody recognizing phosphorylated serine 13 (PS13) within the N-terminus of
mHTTx1. PS13 antibody was purchased from ThermoFisher Scientific. (D) WB showing
the presence of mHTTx1 products (bracket) during the differentiation of NPC expressing
mHTTx1 and mHTTx1 with KD expression of IKKβ. mHTTx1 products were detected
with a new antibody recognizing the C-terminus of mHTTx1. GAPDH was used as
loading control. Left lane labeled as control (C) is lysates from12 days differentiated
neurons, which do not express mHTTx1.
Figure S6. WB analysis of lysates of developing neurons expressing mHTTx1 and
mHTTx1 with KD expression of IKKβ for levels of procaspase-3 and cleaved caspase-3,
probed with anti-caspase-3 antibody (Cell signaling, Danvers, MA). No cleaved products
were observed. Results confirm data in Fig. 5A that KD of IKKβ prevents mHTTx1-
induced procaspase-3 accumulation (rightmost lanes).