Protocol detail

Monitoring Ipa Protein Secretion

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The secretion of Ipa proteins after Congo red induction was carried our as previously described [11 (link)]. The total level of protein expression was revealed via western blot using monoclonal anti-IpaB H16 [57 (link)] and polyclonal anti-IpaH [58 (link)], anti-RNAP α subunit (gift from Prof. Akira Ishihama, Hosei University, Koganai, Tokyo) and anti-λ cI (gift from Prof. Ann Hochschild, Harvard Medical School, Boston). Goat anti-mouse DyLight 800 (Fisher Scientific) or goat anti-rabbit Alexa 680 (Invitrogen) conjugates were used as secondary antibodies. The membranes were then visualized using an Odyssey infrared imaging system (LI-COR Biosciences).

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Shen D.K, & Blocker A.J. (2016). MxiA, MxiC and IpaD Regulate Substrate Selection and Secretion Mode in the T3SS of Shigella flexneri. PLoS ONE, 11(5), e0155141.

Publication 2016

DyLight 800 goat anti-rabbit Alexa 680 Odyssey infrared imaging system

Antibodies Goat Membranes Mouse Protein Rabbit Secretion Subunit Western blot

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Variable analysis

independent variables

Congo red induction

dependent variables

Secretion of Ipa proteins
Total level of protein expression

control variables

Anti-IpaB H16 [57]
Polyclonal anti-IpaH [58]
Anti-RNAP α subunit (gift from Prof. Akira Ishihama, Hosei University, Koganai, Tokyo)
Anti-λ cI (gift from Prof. Ann Hochschild, Harvard Medical School, Boston)
Goat anti-mouse DyLight 800 (Fisher Scientific)
Goat anti-rabbit Alexa 680 (Invitrogen)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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