Validated Antibody Characterization for Microscopy and Immunoblotting

Antibodies used for imaging include: anti-Pyk2 (Santa Cruz Biotechnology, Inc.; cat#: sc-393181), anti-vinculin (Santa Cruz Biotechnology, Inc.; cat#: sc-25336), anti-L-plastin (Santa Cruz Biotechnology, Inc.; cat#: sc-133218), and anti-Tks5 (Proteintech; Cat#: 18976-1-AP) and anti-phalloidin IFluor 647 (Abcam; cat#: ab176759).
Primary antibodies used for Western blot include: anti-Pyk2 (Cell Signaling; cat#: 3292S), anti-vinculin (Santa Cruz Biotechnology, Inc.; cat#: sc-25336), and anti-beta actin (Cell Signaling; cat#: 8457S). Anti-L-plastin was provided by Eric J. Brown, Genentech (Morley et al., 2010 (link); Wang et al., 2010 (link)). Specificity of anti-LPL antibodies has been validated by immunofluorescent microscopy using LPL-deficient cells Supplementary Figure S1 (Morley et al., 2010 (link)); and by immunoblot (Anaya et al., 2021 (link); Joshi et al., 2022 (link)). Specificity of anti-Pyk2, anti-actin and anti-vinculin were confirmed by visualization of single protein band in immunoblots (Supplementary Figure S2).

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Lin X., Krishnamoorthy P., Walker E.C., Joshi H, & Morley S.C. (2023). Expression of non-phosphorylatable S5A-L-plastin exerts phenotypes distinct from L-plastin deficiency during podosome formation and phagocytosis. Frontiers in Cell and Developmental Biology, 11, 1020091.

Publication 2023

anti-Pyk2 anti-vinculin anti-beta actin

Actin Anti antibodies Antibodies Beta actin Cells Immunoblots Immunofluorescent microscopy Phalloidin Plastin Protein Vinculin Western blot

Corresponding Organization : Washington University in St. Louis

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Variable analysis

independent variables

Primary antibodies used for Western blot: anti-Pyk2, anti-vinculin, and anti-beta actin

dependent variables

Antibodies used for imaging: anti-Pyk2, anti-vinculin, anti-L-plastin, anti-Tks5, and anti-phalloidin IFluor 647

control variables

Specificity of anti-LPL antibodies has been validated by immunofluorescent microscopy using LPL-deficient cells (Supplementary Figure S1) and by immunoblot
Specificity of anti-Pyk2, anti-actin and anti-vinculin were confirmed by visualization of single protein band in immunoblots (Supplementary Figure S2)

positive controls

Anti-L-plastin was provided by Eric J. Brown, Genentech (Morley et al., 2010; Wang et al., 2010)

negative controls

LPL-deficient cells (Supplementary Figure S1)

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