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3 protocols using anti c myc agarose

1

Antibody Characterization and Validation

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The primary antibodies used were as follows: rabbit anti-β-tubulin (immunoblot 1:1500; Santa Cruz); mouse anti-ezrin (immunoblot 1:1000, IF 1:300; Santa Cruz); rabbit anti-ezrin (IF 1:300; Cell Signaling); rabbit anti-GFP (immunoblot 1:1000; Santa Cruz); rabbit anti-His-probe (immunoblot 1:1000; Santa Cruz); mouse anti-Myc (immunoblot 1:1500; Santa Cruz); rabbit anti-MYOGEF (immunoblot 1:100); mouse anti-MYOGEF (IF 1:100); mouse anti phosphomyosin light chain 2 (IF 1:500; Cell Signaling). Rabbit and mouse anti-MYOGEF antibodies have been described previously (Wu et al., 2006 (link), 2014a (link)).
Secondary antibodies include Alexa Fluor 594 Goat anti-mouse IgG (IF 1:500; Thermo Fisher Scientific), Alexa Fluor 488 Goat anti-mouse IgG (IF 1:500; Thermo Fisher Scientific), Alexa Fluor 488 Goat anti-rabbit IgG (IF 1:500; Thermo Fisher Scientific), europium-labeled anti-mouse (immunoblot 1:5000; Molecular Devices), and europium-labeled anti-rabbit (immunoblot 1:5000; Molecular Devices).
Protein A-Sepharose beads and anti-c-Myc agarose were purchased from Santa Cruz. Nocodazole and puromycin were purchased from Sigma. The phospholipase C activator m-3M3FBS was purchased from EMD Millipore.
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2

Immunoblotting Protocols Using Diverse Antibodies

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Primary antibodies used were from the following sources: rabbit (Rb) anti-Myc (Cell Signaling, 2278), mouse (Ms) anti-Myc (Cell Signaling, 9B11), Rb anti-actin (Sigma, A2066), Ms anti-FLAG (Sigma F1804), Rb anti-14-3-3 (Santa Cruz, sc-629), Ms anti -Spir-1 (Santa Cruz, sc-517039), Ms anti-Spir-1 (Abcam, ab57463), Rb anti-DDX3 (Cell Signaling, 2635), Rb anti-IKKβ (Cell Signaling, 2684), Rb anti-HA (Sigma, H6908), Ms anti-α-tubulin (Millipore, 05–829), Ms anti-GAPDH (Sigma, G8795), Rb anti-IRF3 (Cell Signaling, 4962), Rb anti-IRF3 (Santa Cruz, SC-9082), Ms anti-COPε (Santa Cruz, sc-133194), Rb anti-phospho-IRF3 Ser396 (Cell Signaling, 4947S) and Rb polyclonal anti-C6 [53 (link)]. For dilutions used for the primary antibodies, see S3 Table. Secondary antibodies used (1:10,000 dilution) were IRDye 680RD-conjugated goat anti-rabbit IgG or anti-mouse IgG and IRDye 800CW-conjugated goat anti-rabbit IgG or anti-mouse IgG (LI-COR).
Reagents used in this study were: anti-c-Myc agarose from Santa Cruz Biotechnology, and monoclonal anti-HA-agarose, clone HA-7, ANTI-FLAG M2 affinity gel and Poly-D-lysine hydrobromide (all from Sigma Aldrich). Human IFNα, human TNF-α and mouse IL-1β were from Peprotech, HMW poly(I:C) and puromycin were from InvivoGen, and doxycycline was from Melford.
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3

Antibody Characterization for Immunoblot

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Primary antibodies used were from the following sources: rabbit (Rb) anti-Myc (Cell Signaling, 2278), mouse (Ms) anti-Myc (Cell Signaling, 9B11), Rb anti-Actin (Sigma, A2066), Ms anti-FLAG (Sigma F1804), Rb anti-14-3-3 (Santa Cruz, sc-629), Ms anti -Spir-1 (Santa Cruz, sc-517039), Ms anti-Spir-1 (Abcam, ab57463), Rb anti-DDX3 (Cell Signaling, 2635), Rb anti-IKKb (Cell Signaling, 2684), Rb anti-HA (Sigma, H6908), Ms anti-a-Tubulin (Millipore, 05-829), Ms anti-GAPDH (Sigma, G8795), Rb anti-IRF3 (Cell Signaling, 4962), Ms anti-COPe (Santa Cruz, sc-133194), Rb anti-phospho-IRF3 Ser396 (Cell Signaling, 4947S) and Rb polyclonal anti-C6 (53) . For dilutions used for the primary antibodies, see reagents table. Secondary antibodies used (1:10,000 dilution) were IRDye 680RDconjugated goat anti-rabbit IgG or anti-mouse IgG and IRDye 800CW-conjugated goat anti-rabbit IgG or anti-mouse IgG (LI-COR).
Reagents used in this study were: Anti-c-Myc Agarose from Santa Cruz Biotechnology, and monoclonal Anti-HA-Agarose, clone HA-7, ANTI-FLAG M2 Affinity Gel and Poly-D-lysine hydrobromide (all from Sigma Aldrich). Human IFNα, human TNF-α and mouse IL-1β were from Peprotech, HMW poly(I:C) and puromycin were from InvivoGen, and doxycycline was from Melford.
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