Western Blot Analysis of DRG Neuron Proteins

DRG neurons transduced with Sarm1, IP₃R1, or control shRNA for 6–7 d were lysed with nonionic detergent. Lysates were separated by 3–8% Tris-Acetate SDS-PAGE for IP₃R1 and by 4–12% Bis-Tris SDS-Page for Sarm1 (Thermo Fisher Scientific) and probed with mouse anti-Sarm1 (1:10,000; Chen et al., 2011 (link)), rabbit anti-IP₃R1 (1:1,000; Thermo Fisher Scientific; PA1-901), and rabbit anti–pan-actin (1:1,000; Cell Signaling; 8456S). Bands were visualized with secondary antibodies conjugated to HRP (1:10,000; Bio-Rad; 1721019, 1706516) and SuperSignal chemiluminescent substrates signal (Thermo Fisher Scientific). Blots were imaged using the AI600 Chemiluminescent Imager (GE Healthcare).

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Li Y., Pazyra-Murphy M.F., Avizonis D., de Sá Tavares Russo M., Tang S., Chen C.Y., Hsueh Y.P., Bergholz J.S., Jiang T., Zhao J.J., Zhu J., Ko K.W., Milbrandt J., DiAntonio A, & Segal R.A. (2021). Sarm1 activation produces cADPR to increase intra-axonal Ca++ and promote axon degeneration in PIPN. The Journal of Cell Biology, 221(2), e202106080.

Publication 2021

PA1-901 rabbit anti–pan-actin SuperSignal chemiluminescent substrates signal AI600 Chemiluminescent Imager

Acetate Actin Antibodies Bis tris Detergent Ip3r1 Mouse Neurons Rabbit Sarm1 Sds page Shrna Tris

Corresponding Organization :

Other organizations : Dana-Farber Cancer Institute, Harvard University, McGill University, Institute of Molecular Biology, Academia Sinica, Broad Institute, Massachusetts Institute of Technology, Washington University in St. Louis, Center for Neuro-Oncology

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Variable analysis

independent variables

Sarm1
IP3R1
Control shRNA

dependent variables

Sarm1 protein expression
IP3R1 protein expression

control variables

Incubation time (6-7 days)
Cell type (DRG neurons)
Lysis method (nonionic detergent)
SDS-PAGE conditions (3-8% Tris-Acetate for IP3R1, 4-12% Bis-Tris for Sarm1)
Primary antibodies (mouse anti-Sarm1, rabbit anti-IP3R1, rabbit anti-pan-actin)
Secondary antibodies (HRP-conjugated)
Chemiluminescent detection (SuperSignal)

positive controls

None specified

negative controls

Control shRNA

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