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pserine  (OriGene)


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    OriGene pserine
    FIGURE 7 The effect of acute ethanol-exposure on pAtg4B and microtubule associated protein 1 light chain-3B (LC3) I and II expression in macrophages. (A) WT-BMDM were exposed to vehicle or ethanol ± LPS. Atg4B was immunoprecipitated (IP) from whole-cell lysates of WT-BMDM using an anti-Atg4B antibody followed by IB analysis of <t>pSerine</t> and total Atg4B. IP with IgG control antibody was used as a negative control. (B) Western blot analysis of total Atg4B in the whole cell lysate used as input for the Atg4B IP. (C) SIRT2KO-BMDM were exposed to ethanol ± LPS. IP for Atg4B from whole-cell lysates of SIRT2KO-BMDM using an anti-Atg4B antibody followed by IB analysis of pSerine and total Atg4B. IP with IgG control antibody was used as a negative control. (D) Western blot analysis of total Atg4B in the whole cell lysate used as input for the Atg4B IP. (E) LC3-I and LC3-II expression were analyzed by western blot in WT-BMDM exposed to vehicle or ethanol ± LPS. Western blot image quantification of ethanol vs. vehicle-expose WT-BMDM± LPS, showing LC3-I in (F) and LC3-II in (G), normalized to vehicle control (Vehicle-LPS) (n = 4 blots; * p < 0.05). (H) Western blot analysis of LC3-I and II expression in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS. Western blot image quantification in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS for LC3-I in (I) and LC3-II in (J), normalized to ethanol control (Ethanol-LPS) (n = 4 blots; * p < 0.05). (K). Western blot analysis of LC3-I and II expression of Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS. Western blot image quantification of Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS for LC3-I in (L) and LC3-II in (M) normalized to ethanol control (Ethanol-LPS) (n = 3 blots; * p < 0.05).
    Pserine, supplied by OriGene, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pserine/Phosphoserine+(incl%2E+pos%2E+control)+Mouse+Monoclonal+Antibody/pm36865524-57-163-164
    Average 91 stars, based on 2 article reviews
    pserine - by Bioz Stars, 2026-10
    91/100 stars

    Images

    1) Product Images from "SIRT2-PFKP interaction dysregulates phagocytosis in macrophages with acute ethanol-exposure."

    Article Title: SIRT2-PFKP interaction dysregulates phagocytosis in macrophages with acute ethanol-exposure.

    Journal: Frontiers in immunology

    doi: 10.3389/fimmu.2022.1079962

    FIGURE 7 The effect of acute ethanol-exposure on pAtg4B and microtubule associated protein 1 light chain-3B (LC3) I and II expression in macrophages. (A) WT-BMDM were exposed to vehicle or ethanol ± LPS. Atg4B was immunoprecipitated (IP) from whole-cell lysates of WT-BMDM using an anti-Atg4B antibody followed by IB analysis of pSerine and total Atg4B. IP with IgG control antibody was used as a negative control. (B) Western blot analysis of total Atg4B in the whole cell lysate used as input for the Atg4B IP. (C) SIRT2KO-BMDM were exposed to ethanol ± LPS. IP for Atg4B from whole-cell lysates of SIRT2KO-BMDM using an anti-Atg4B antibody followed by IB analysis of pSerine and total Atg4B. IP with IgG control antibody was used as a negative control. (D) Western blot analysis of total Atg4B in the whole cell lysate used as input for the Atg4B IP. (E) LC3-I and LC3-II expression were analyzed by western blot in WT-BMDM exposed to vehicle or ethanol ± LPS. Western blot image quantification of ethanol vs. vehicle-expose WT-BMDM± LPS, showing LC3-I in (F) and LC3-II in (G), normalized to vehicle control (Vehicle-LPS) (n = 4 blots; * p < 0.05). (H) Western blot analysis of LC3-I and II expression in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS. Western blot image quantification in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS for LC3-I in (I) and LC3-II in (J), normalized to ethanol control (Ethanol-LPS) (n = 4 blots; * p < 0.05). (K). Western blot analysis of LC3-I and II expression of Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS. Western blot image quantification of Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS for LC3-I in (L) and LC3-II in (M) normalized to ethanol control (Ethanol-LPS) (n = 3 blots; * p < 0.05).
    Figure Legend Snippet: FIGURE 7 The effect of acute ethanol-exposure on pAtg4B and microtubule associated protein 1 light chain-3B (LC3) I and II expression in macrophages. (A) WT-BMDM were exposed to vehicle or ethanol ± LPS. Atg4B was immunoprecipitated (IP) from whole-cell lysates of WT-BMDM using an anti-Atg4B antibody followed by IB analysis of pSerine and total Atg4B. IP with IgG control antibody was used as a negative control. (B) Western blot analysis of total Atg4B in the whole cell lysate used as input for the Atg4B IP. (C) SIRT2KO-BMDM were exposed to ethanol ± LPS. IP for Atg4B from whole-cell lysates of SIRT2KO-BMDM using an anti-Atg4B antibody followed by IB analysis of pSerine and total Atg4B. IP with IgG control antibody was used as a negative control. (D) Western blot analysis of total Atg4B in the whole cell lysate used as input for the Atg4B IP. (E) LC3-I and LC3-II expression were analyzed by western blot in WT-BMDM exposed to vehicle or ethanol ± LPS. Western blot image quantification of ethanol vs. vehicle-expose WT-BMDM± LPS, showing LC3-I in (F) and LC3-II in (G), normalized to vehicle control (Vehicle-LPS) (n = 4 blots; * p < 0.05). (H) Western blot analysis of LC3-I and II expression in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS. Western blot image quantification in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS for LC3-I in (I) and LC3-II in (J), normalized to ethanol control (Ethanol-LPS) (n = 4 blots; * p < 0.05). (K). Western blot analysis of LC3-I and II expression of Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS. Western blot image quantification of Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS for LC3-I in (L) and LC3-II in (M) normalized to ethanol control (Ethanol-LPS) (n = 3 blots; * p < 0.05).

    Techniques Used: Expressing, Immunoprecipitation, Control, Negative Control, Western Blot

    Related Articles

    Western Blot:

    Article Title: SIRT2-PFKP interaction dysregulates phagocytosis in macrophages with acute ethanol-exposure.
    Article Snippet: .. Antibodies and reagents used for western blot and immunocytochemistry PFKP (Cell signaling Technology, Danvers, MA, CAT# 8164S), LC3 (Cell signaling Technology, Danvers, MA, CAT# E5Q2K: western blot), LC3 (Novus Biologicals, Centennial, Colorado, CAT# NC100-2220: immunocytochemistry), SIRT2 (Cell signaling Technology, Danvers, MA, CAT# D4050), Atg4B (Cell signaling Technology, Danvers, MA, CAT# 13507S), Rubicon (Cell signaling Technology, Danvers, MA, CAT# 8465S), Beclin-1 (Cell signaling Technology, CAT# 3495S), PFKM (Abcam, Waltham, Boston, USA,CAT#ab154804), VPS34 (Abcam, Waltham, Boston, CAT# ab124905), PFKL (Santa Cruz Biotechnology, Dallas, Texas, USA, CAT# sc393713), F4/80 (Invitrogen, Rockford, Illinois, USA, CAT# MA1-91124), Anti-rabbit Alexa Fluor 488 (Invitrogen, Rockford, Illinois, USA, CAT# A21206), Anti-rat Alexa Fluor 594 (Invitrogen, Rockford, Illinois, USA, CAT# A21209), Antirabbit Alexa Fluor 647 (Invitrogen, Rockford, Illinois, USA, CAT# A21245), Anti rabbit IgG control (Cell signaling Technology, Danvers, MA, CAT# 2729S), anti-mouse IgG, HRP-linked antibody (Cell signaling Technology, Danvers, MA, CAT# 7076), Anti-rabbit IgG, HRP-linked antibody (Cell signaling Technology, CAT# 7074), Acetyl lysine (Novus Biologicals, Centennial, CO, USA, CAT# NB 100-74339) pSerine (Origene, Rockville, MD, USA, CAT# AM00114PUN), Anti- Turbo GFP (tGFP) antibody (Origene, Rockville, MD, CAT# TA150041), DDK antibody (Origene, Rockville, MD, CAT# TA50011-100), Anti-Ubiquitin antibody (EMD Millipore, Burlington, Massachusetts, USA, CAT#5-944), Biotinylated anti-SIRT2 antibody (R&D system, Minneapolis, MN, USA, CAT# BAF4358), CPA (Novus Biologicals, CAT# NBP1-30993), Beta-actin(Abcam, Waltham, Boston, CAT# ab8226), ECL (Bio-Rad, Hercules, CA, USA, CAT# 1705061). .. ChromoTek TurboGFP-Trap Magnetic Agarose (Proteintech Group, Inc, Rosemont, IL, CAT# tbtma-10), Magnetic-TUBEs (Life Sensors, Malvern, PA, USA, CAT# UM501M), Zymosan A pHrodo red bio-particles (Invitrogen, Waltham, MA, USA, CAT# P35364), Vybrant phagocytosis assay (Thermo Fisher Scientific, Waltham, MA, USA, CAT# V-6694).

    Article Title: SIRT2-PFKP interaction dysregulates phagocytosis in macrophages with acute ethanol-exposure
    Article Snippet: .. PFKP ( Cell signaling Technology, Danvers, MA, CAT# 8164S), LC3 (Cell signaling Technology, Danvers, MA, CAT# E5Q2K: western blot), LC3 (Novus Biologicals, Centennial, Colorado, CAT# NC100-2220: immunocytochemistry), SIRT2 (Cell signaling Technology, Danvers, MA, CAT# D4050), Atg4B (Cell signaling Technology, Danvers, MA, CAT# 13507S), Rubicon (Cell signaling Technology, Danvers, MA, CAT# 8465S), Beclin-1 (Cell signaling Technology, CAT# 3495S), PFKM (Abcam, Waltham, Boston, USA,CAT#ab154804), VPS34 (Abcam, Waltham, Boston, CAT# ab124905), PFKL (Santa Cruz Biotechnology, Dallas, Texas, USA, CAT# sc-393713), F4/80 (Invitrogen, Rockford, Illinois, USA, CAT# MA1-91124), Anti-rabbit Alexa Fluor 488 (Invitrogen, Rockford, Illinois, USA, CAT# A21206), Anti-rat Alexa Fluor 594 (Invitrogen, Rockford, Illinois, USA, CAT# A21209), Anti-rabbit Alexa Fluor 647 (Invitrogen, Rockford, Illinois, USA, CAT# A21245), Anti rabbit IgG control (Cell signaling Technology, Danvers, MA, CAT# 2729S), anti-mouse IgG, HRP-linked antibody (Cell signaling Technology, Danvers, MA, CAT# 7076), Anti-rabbit IgG, HRP-linked antibody (Cell signaling Technology, CAT# 7074), Acetyl lysine (Novus Biologicals, Centennial, CO, USA, CAT# NB 100-74339) pSerine (Origene, Rockville, MD, USA, CAT# AM00114PU-N), Anti- Turbo GFP (tGFP) antibody (Origene, Rockville, MD, CAT# TA150041), DDK antibody (Origene, Rockville, MD, CAT# TA50011-100), Anti-Ubiquitin antibody (EMD Millipore, Burlington, Massachusetts, USA, CAT#5-944), Biotinylated anti-SIRT2 antibody (R&D system, Minneapolis, MN, USA, CAT# BAF4358), CPA (Novus Biologicals, CAT# NBP1-30993), Beta-actin(Abcam, Waltham, Boston, CAT# ab8226), ECL (Bio-Rad, Hercules, CA, USA, CAT# 1705061). .. ChromoTek TurboGFP-Trap Magnetic Agarose (Proteintech Group, Inc, Rosemont, IL, CAT# tbtma-10), Magnetic-TUBEs (Life Sensors, Malvern, PA, USA, CAT# UM501M), Zymosan A pHrodo red bio-particles (Invitrogen, Waltham, MA, USA, CAT# P35364), Vybrant phagocytosis assay (Thermo Fisher Scientific, Waltham, MA, USA, CAT# V-6694).

    Immunocytochemistry:

    Article Title: SIRT2-PFKP interaction dysregulates phagocytosis in macrophages with acute ethanol-exposure.
    Article Snippet: .. Antibodies and reagents used for western blot and immunocytochemistry PFKP (Cell signaling Technology, Danvers, MA, CAT# 8164S), LC3 (Cell signaling Technology, Danvers, MA, CAT# E5Q2K: western blot), LC3 (Novus Biologicals, Centennial, Colorado, CAT# NC100-2220: immunocytochemistry), SIRT2 (Cell signaling Technology, Danvers, MA, CAT# D4050), Atg4B (Cell signaling Technology, Danvers, MA, CAT# 13507S), Rubicon (Cell signaling Technology, Danvers, MA, CAT# 8465S), Beclin-1 (Cell signaling Technology, CAT# 3495S), PFKM (Abcam, Waltham, Boston, USA,CAT#ab154804), VPS34 (Abcam, Waltham, Boston, CAT# ab124905), PFKL (Santa Cruz Biotechnology, Dallas, Texas, USA, CAT# sc393713), F4/80 (Invitrogen, Rockford, Illinois, USA, CAT# MA1-91124), Anti-rabbit Alexa Fluor 488 (Invitrogen, Rockford, Illinois, USA, CAT# A21206), Anti-rat Alexa Fluor 594 (Invitrogen, Rockford, Illinois, USA, CAT# A21209), Antirabbit Alexa Fluor 647 (Invitrogen, Rockford, Illinois, USA, CAT# A21245), Anti rabbit IgG control (Cell signaling Technology, Danvers, MA, CAT# 2729S), anti-mouse IgG, HRP-linked antibody (Cell signaling Technology, Danvers, MA, CAT# 7076), Anti-rabbit IgG, HRP-linked antibody (Cell signaling Technology, CAT# 7074), Acetyl lysine (Novus Biologicals, Centennial, CO, USA, CAT# NB 100-74339) pSerine (Origene, Rockville, MD, USA, CAT# AM00114PUN), Anti- Turbo GFP (tGFP) antibody (Origene, Rockville, MD, CAT# TA150041), DDK antibody (Origene, Rockville, MD, CAT# TA50011-100), Anti-Ubiquitin antibody (EMD Millipore, Burlington, Massachusetts, USA, CAT#5-944), Biotinylated anti-SIRT2 antibody (R&D system, Minneapolis, MN, USA, CAT# BAF4358), CPA (Novus Biologicals, CAT# NBP1-30993), Beta-actin(Abcam, Waltham, Boston, CAT# ab8226), ECL (Bio-Rad, Hercules, CA, USA, CAT# 1705061). .. ChromoTek TurboGFP-Trap Magnetic Agarose (Proteintech Group, Inc, Rosemont, IL, CAT# tbtma-10), Magnetic-TUBEs (Life Sensors, Malvern, PA, USA, CAT# UM501M), Zymosan A pHrodo red bio-particles (Invitrogen, Waltham, MA, USA, CAT# P35364), Vybrant phagocytosis assay (Thermo Fisher Scientific, Waltham, MA, USA, CAT# V-6694).

    Article Title: SIRT2-PFKP interaction dysregulates phagocytosis in macrophages with acute ethanol-exposure
    Article Snippet: .. PFKP ( Cell signaling Technology, Danvers, MA, CAT# 8164S), LC3 (Cell signaling Technology, Danvers, MA, CAT# E5Q2K: western blot), LC3 (Novus Biologicals, Centennial, Colorado, CAT# NC100-2220: immunocytochemistry), SIRT2 (Cell signaling Technology, Danvers, MA, CAT# D4050), Atg4B (Cell signaling Technology, Danvers, MA, CAT# 13507S), Rubicon (Cell signaling Technology, Danvers, MA, CAT# 8465S), Beclin-1 (Cell signaling Technology, CAT# 3495S), PFKM (Abcam, Waltham, Boston, USA,CAT#ab154804), VPS34 (Abcam, Waltham, Boston, CAT# ab124905), PFKL (Santa Cruz Biotechnology, Dallas, Texas, USA, CAT# sc-393713), F4/80 (Invitrogen, Rockford, Illinois, USA, CAT# MA1-91124), Anti-rabbit Alexa Fluor 488 (Invitrogen, Rockford, Illinois, USA, CAT# A21206), Anti-rat Alexa Fluor 594 (Invitrogen, Rockford, Illinois, USA, CAT# A21209), Anti-rabbit Alexa Fluor 647 (Invitrogen, Rockford, Illinois, USA, CAT# A21245), Anti rabbit IgG control (Cell signaling Technology, Danvers, MA, CAT# 2729S), anti-mouse IgG, HRP-linked antibody (Cell signaling Technology, Danvers, MA, CAT# 7076), Anti-rabbit IgG, HRP-linked antibody (Cell signaling Technology, CAT# 7074), Acetyl lysine (Novus Biologicals, Centennial, CO, USA, CAT# NB 100-74339) pSerine (Origene, Rockville, MD, USA, CAT# AM00114PU-N), Anti- Turbo GFP (tGFP) antibody (Origene, Rockville, MD, CAT# TA150041), DDK antibody (Origene, Rockville, MD, CAT# TA50011-100), Anti-Ubiquitin antibody (EMD Millipore, Burlington, Massachusetts, USA, CAT#5-944), Biotinylated anti-SIRT2 antibody (R&D system, Minneapolis, MN, USA, CAT# BAF4358), CPA (Novus Biologicals, CAT# NBP1-30993), Beta-actin(Abcam, Waltham, Boston, CAT# ab8226), ECL (Bio-Rad, Hercules, CA, USA, CAT# 1705061). .. ChromoTek TurboGFP-Trap Magnetic Agarose (Proteintech Group, Inc, Rosemont, IL, CAT# tbtma-10), Magnetic-TUBEs (Life Sensors, Malvern, PA, USA, CAT# UM501M), Zymosan A pHrodo red bio-particles (Invitrogen, Waltham, MA, USA, CAT# P35364), Vybrant phagocytosis assay (Thermo Fisher Scientific, Waltham, MA, USA, CAT# V-6694).

    Control:

    Article Title: SIRT2-PFKP interaction dysregulates phagocytosis in macrophages with acute ethanol-exposure.
    Article Snippet: .. Antibodies and reagents used for western blot and immunocytochemistry PFKP (Cell signaling Technology, Danvers, MA, CAT# 8164S), LC3 (Cell signaling Technology, Danvers, MA, CAT# E5Q2K: western blot), LC3 (Novus Biologicals, Centennial, Colorado, CAT# NC100-2220: immunocytochemistry), SIRT2 (Cell signaling Technology, Danvers, MA, CAT# D4050), Atg4B (Cell signaling Technology, Danvers, MA, CAT# 13507S), Rubicon (Cell signaling Technology, Danvers, MA, CAT# 8465S), Beclin-1 (Cell signaling Technology, CAT# 3495S), PFKM (Abcam, Waltham, Boston, USA,CAT#ab154804), VPS34 (Abcam, Waltham, Boston, CAT# ab124905), PFKL (Santa Cruz Biotechnology, Dallas, Texas, USA, CAT# sc393713), F4/80 (Invitrogen, Rockford, Illinois, USA, CAT# MA1-91124), Anti-rabbit Alexa Fluor 488 (Invitrogen, Rockford, Illinois, USA, CAT# A21206), Anti-rat Alexa Fluor 594 (Invitrogen, Rockford, Illinois, USA, CAT# A21209), Antirabbit Alexa Fluor 647 (Invitrogen, Rockford, Illinois, USA, CAT# A21245), Anti rabbit IgG control (Cell signaling Technology, Danvers, MA, CAT# 2729S), anti-mouse IgG, HRP-linked antibody (Cell signaling Technology, Danvers, MA, CAT# 7076), Anti-rabbit IgG, HRP-linked antibody (Cell signaling Technology, CAT# 7074), Acetyl lysine (Novus Biologicals, Centennial, CO, USA, CAT# NB 100-74339) pSerine (Origene, Rockville, MD, USA, CAT# AM00114PUN), Anti- Turbo GFP (tGFP) antibody (Origene, Rockville, MD, CAT# TA150041), DDK antibody (Origene, Rockville, MD, CAT# TA50011-100), Anti-Ubiquitin antibody (EMD Millipore, Burlington, Massachusetts, USA, CAT#5-944), Biotinylated anti-SIRT2 antibody (R&D system, Minneapolis, MN, USA, CAT# BAF4358), CPA (Novus Biologicals, CAT# NBP1-30993), Beta-actin(Abcam, Waltham, Boston, CAT# ab8226), ECL (Bio-Rad, Hercules, CA, USA, CAT# 1705061). .. ChromoTek TurboGFP-Trap Magnetic Agarose (Proteintech Group, Inc, Rosemont, IL, CAT# tbtma-10), Magnetic-TUBEs (Life Sensors, Malvern, PA, USA, CAT# UM501M), Zymosan A pHrodo red bio-particles (Invitrogen, Waltham, MA, USA, CAT# P35364), Vybrant phagocytosis assay (Thermo Fisher Scientific, Waltham, MA, USA, CAT# V-6694).

    Article Title: SIRT2-PFKP interaction dysregulates phagocytosis in macrophages with acute ethanol-exposure
    Article Snippet: .. PFKP ( Cell signaling Technology, Danvers, MA, CAT# 8164S), LC3 (Cell signaling Technology, Danvers, MA, CAT# E5Q2K: western blot), LC3 (Novus Biologicals, Centennial, Colorado, CAT# NC100-2220: immunocytochemistry), SIRT2 (Cell signaling Technology, Danvers, MA, CAT# D4050), Atg4B (Cell signaling Technology, Danvers, MA, CAT# 13507S), Rubicon (Cell signaling Technology, Danvers, MA, CAT# 8465S), Beclin-1 (Cell signaling Technology, CAT# 3495S), PFKM (Abcam, Waltham, Boston, USA,CAT#ab154804), VPS34 (Abcam, Waltham, Boston, CAT# ab124905), PFKL (Santa Cruz Biotechnology, Dallas, Texas, USA, CAT# sc-393713), F4/80 (Invitrogen, Rockford, Illinois, USA, CAT# MA1-91124), Anti-rabbit Alexa Fluor 488 (Invitrogen, Rockford, Illinois, USA, CAT# A21206), Anti-rat Alexa Fluor 594 (Invitrogen, Rockford, Illinois, USA, CAT# A21209), Anti-rabbit Alexa Fluor 647 (Invitrogen, Rockford, Illinois, USA, CAT# A21245), Anti rabbit IgG control (Cell signaling Technology, Danvers, MA, CAT# 2729S), anti-mouse IgG, HRP-linked antibody (Cell signaling Technology, Danvers, MA, CAT# 7076), Anti-rabbit IgG, HRP-linked antibody (Cell signaling Technology, CAT# 7074), Acetyl lysine (Novus Biologicals, Centennial, CO, USA, CAT# NB 100-74339) pSerine (Origene, Rockville, MD, USA, CAT# AM00114PU-N), Anti- Turbo GFP (tGFP) antibody (Origene, Rockville, MD, CAT# TA150041), DDK antibody (Origene, Rockville, MD, CAT# TA50011-100), Anti-Ubiquitin antibody (EMD Millipore, Burlington, Massachusetts, USA, CAT#5-944), Biotinylated anti-SIRT2 antibody (R&D system, Minneapolis, MN, USA, CAT# BAF4358), CPA (Novus Biologicals, CAT# NBP1-30993), Beta-actin(Abcam, Waltham, Boston, CAT# ab8226), ECL (Bio-Rad, Hercules, CA, USA, CAT# 1705061). .. ChromoTek TurboGFP-Trap Magnetic Agarose (Proteintech Group, Inc, Rosemont, IL, CAT# tbtma-10), Magnetic-TUBEs (Life Sensors, Malvern, PA, USA, CAT# UM501M), Zymosan A pHrodo red bio-particles (Invitrogen, Waltham, MA, USA, CAT# P35364), Vybrant phagocytosis assay (Thermo Fisher Scientific, Waltham, MA, USA, CAT# V-6694).



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    FIGURE 7 The effect of acute ethanol-exposure on pAtg4B and microtubule associated protein 1 light chain-3B (LC3) I and II expression in macrophages. (A) WT-BMDM were exposed to vehicle or ethanol ± LPS. Atg4B was immunoprecipitated (IP) from whole-cell lysates of WT-BMDM using an anti-Atg4B antibody followed by IB analysis of <t>pSerine</t> and total Atg4B. IP with IgG control antibody was used as a negative control. (B) Western blot analysis of total Atg4B in the whole cell lysate used as input for the Atg4B IP. (C) SIRT2KO-BMDM were exposed to ethanol ± LPS. IP for Atg4B from whole-cell lysates of SIRT2KO-BMDM using an anti-Atg4B antibody followed by IB analysis of pSerine and total Atg4B. IP with IgG control antibody was used as a negative control. (D) Western blot analysis of total Atg4B in the whole cell lysate used as input for the Atg4B IP. (E) LC3-I and LC3-II expression were analyzed by western blot in WT-BMDM exposed to vehicle or ethanol ± LPS. Western blot image quantification of ethanol vs. vehicle-expose WT-BMDM± LPS, showing LC3-I in (F) and LC3-II in (G), normalized to vehicle control (Vehicle-LPS) (n = 4 blots; * p < 0.05). (H) Western blot analysis of LC3-I and II expression in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS. Western blot image quantification in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS for LC3-I in (I) and LC3-II in (J), normalized to ethanol control (Ethanol-LPS) (n = 4 blots; * p < 0.05). (K). Western blot analysis of LC3-I and II expression of Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS. Western blot image quantification of Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS for LC3-I in (L) and LC3-II in (M) normalized to ethanol control (Ethanol-LPS) (n = 3 blots; * p < 0.05).
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    FIGURE 7 The effect of acute ethanol-exposure on pAtg4B and microtubule associated protein 1 light chain-3B (LC3) I and II expression in macrophages. (A) WT-BMDM were exposed to vehicle or ethanol ± LPS. Atg4B was immunoprecipitated (IP) from whole-cell lysates of WT-BMDM using an anti-Atg4B antibody followed by IB analysis of <t>pSerine</t> and total Atg4B. IP with IgG control antibody was used as a negative control. (B) Western blot analysis of total Atg4B in the whole cell lysate used as input for the Atg4B IP. (C) SIRT2KO-BMDM were exposed to ethanol ± LPS. IP for Atg4B from whole-cell lysates of SIRT2KO-BMDM using an anti-Atg4B antibody followed by IB analysis of pSerine and total Atg4B. IP with IgG control antibody was used as a negative control. (D) Western blot analysis of total Atg4B in the whole cell lysate used as input for the Atg4B IP. (E) LC3-I and LC3-II expression were analyzed by western blot in WT-BMDM exposed to vehicle or ethanol ± LPS. Western blot image quantification of ethanol vs. vehicle-expose WT-BMDM± LPS, showing LC3-I in (F) and LC3-II in (G), normalized to vehicle control (Vehicle-LPS) (n = 4 blots; * p < 0.05). (H) Western blot analysis of LC3-I and II expression in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS. Western blot image quantification in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS for LC3-I in (I) and LC3-II in (J), normalized to ethanol control (Ethanol-LPS) (n = 4 blots; * p < 0.05). (K). Western blot analysis of LC3-I and II expression of Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS. Western blot image quantification of Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS for LC3-I in (L) and LC3-II in (M) normalized to ethanol control (Ethanol-LPS) (n = 3 blots; * p < 0.05).
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    FIGURE 7 The effect of acute ethanol-exposure on pAtg4B and microtubule associated protein 1 light chain-3B (LC3) I and II expression in macrophages. (A) WT-BMDM were exposed to vehicle or ethanol ± LPS. Atg4B was immunoprecipitated (IP) from whole-cell lysates of WT-BMDM using an anti-Atg4B antibody followed by IB analysis of <t>pSerine</t> and total Atg4B. IP with IgG control antibody was used as a negative control. (B) Western blot analysis of total Atg4B in the whole cell lysate used as input for the Atg4B IP. (C) SIRT2KO-BMDM were exposed to ethanol ± LPS. IP for Atg4B from whole-cell lysates of SIRT2KO-BMDM using an anti-Atg4B antibody followed by IB analysis of pSerine and total Atg4B. IP with IgG control antibody was used as a negative control. (D) Western blot analysis of total Atg4B in the whole cell lysate used as input for the Atg4B IP. (E) LC3-I and LC3-II expression were analyzed by western blot in WT-BMDM exposed to vehicle or ethanol ± LPS. Western blot image quantification of ethanol vs. vehicle-expose WT-BMDM± LPS, showing LC3-I in (F) and LC3-II in (G), normalized to vehicle control (Vehicle-LPS) (n = 4 blots; * p < 0.05). (H) Western blot analysis of LC3-I and II expression in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS. Western blot image quantification in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS for LC3-I in (I) and LC3-II in (J), normalized to ethanol control (Ethanol-LPS) (n = 4 blots; * p < 0.05). (K). Western blot analysis of LC3-I and II expression of Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS. Western blot image quantification of Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS for LC3-I in (L) and LC3-II in (M) normalized to ethanol control (Ethanol-LPS) (n = 3 blots; * p < 0.05).
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    FIGURE 7 The effect of acute ethanol-exposure on pAtg4B and microtubule associated protein 1 light chain-3B (LC3) I and II expression in macrophages. (A) WT-BMDM were exposed to vehicle or ethanol ± LPS. Atg4B was immunoprecipitated (IP) from whole-cell lysates of WT-BMDM using an anti-Atg4B antibody followed by IB analysis of <t>pSerine</t> and total Atg4B. IP with IgG control antibody was used as a negative control. (B) Western blot analysis of total Atg4B in the whole cell lysate used as input for the Atg4B IP. (C) SIRT2KO-BMDM were exposed to ethanol ± LPS. IP for Atg4B from whole-cell lysates of SIRT2KO-BMDM using an anti-Atg4B antibody followed by IB analysis of pSerine and total Atg4B. IP with IgG control antibody was used as a negative control. (D) Western blot analysis of total Atg4B in the whole cell lysate used as input for the Atg4B IP. (E) LC3-I and LC3-II expression were analyzed by western blot in WT-BMDM exposed to vehicle or ethanol ± LPS. Western blot image quantification of ethanol vs. vehicle-expose WT-BMDM± LPS, showing LC3-I in (F) and LC3-II in (G), normalized to vehicle control (Vehicle-LPS) (n = 4 blots; * p < 0.05). (H) Western blot analysis of LC3-I and II expression in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS. Western blot image quantification in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS for LC3-I in (I) and LC3-II in (J), normalized to ethanol control (Ethanol-LPS) (n = 4 blots; * p < 0.05). (K). Western blot analysis of LC3-I and II expression of Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS. Western blot image quantification of Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS for LC3-I in (L) and LC3-II in (M) normalized to ethanol control (Ethanol-LPS) (n = 3 blots; * p < 0.05).
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    FIGURE 7 The effect of acute ethanol-exposure on pAtg4B and microtubule associated protein 1 light chain-3B (LC3) I and II expression in macrophages. (A) WT-BMDM were exposed to vehicle or ethanol ± LPS. Atg4B was immunoprecipitated (IP) from whole-cell lysates of WT-BMDM using an anti-Atg4B antibody followed by IB analysis of <t>pSerine</t> and total Atg4B. IP with IgG control antibody was used as a negative control. (B) Western blot analysis of total Atg4B in the whole cell lysate used as input for the Atg4B IP. (C) SIRT2KO-BMDM were exposed to ethanol ± LPS. IP for Atg4B from whole-cell lysates of SIRT2KO-BMDM using an anti-Atg4B antibody followed by IB analysis of pSerine and total Atg4B. IP with IgG control antibody was used as a negative control. (D) Western blot analysis of total Atg4B in the whole cell lysate used as input for the Atg4B IP. (E) LC3-I and LC3-II expression were analyzed by western blot in WT-BMDM exposed to vehicle or ethanol ± LPS. Western blot image quantification of ethanol vs. vehicle-expose WT-BMDM± LPS, showing LC3-I in (F) and LC3-II in (G), normalized to vehicle control (Vehicle-LPS) (n = 4 blots; * p < 0.05). (H) Western blot analysis of LC3-I and II expression in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS. Western blot image quantification in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS for LC3-I in (I) and LC3-II in (J), normalized to ethanol control (Ethanol-LPS) (n = 4 blots; * p < 0.05). (K). Western blot analysis of LC3-I and II expression of Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS. Western blot image quantification of Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS for LC3-I in (L) and LC3-II in (M) normalized to ethanol control (Ethanol-LPS) (n = 3 blots; * p < 0.05).
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    FIGURE 7 The effect of acute ethanol-exposure on pAtg4B and microtubule associated protein 1 light chain-3B (LC3) I and II expression in macrophages. (A) WT-BMDM were exposed to vehicle or ethanol ± LPS. Atg4B was immunoprecipitated (IP) from whole-cell lysates of WT-BMDM using an anti-Atg4B antibody followed by IB analysis of <t>pSerine</t> and total Atg4B. IP with IgG control antibody was used as a negative control. (B) Western blot analysis of total Atg4B in the whole cell lysate used as input for the Atg4B IP. (C) SIRT2KO-BMDM were exposed to ethanol ± LPS. IP for Atg4B from whole-cell lysates of SIRT2KO-BMDM using an anti-Atg4B antibody followed by IB analysis of pSerine and total Atg4B. IP with IgG control antibody was used as a negative control. (D) Western blot analysis of total Atg4B in the whole cell lysate used as input for the Atg4B IP. (E) LC3-I and LC3-II expression were analyzed by western blot in WT-BMDM exposed to vehicle or ethanol ± LPS. Western blot image quantification of ethanol vs. vehicle-expose WT-BMDM± LPS, showing LC3-I in (F) and LC3-II in (G), normalized to vehicle control (Vehicle-LPS) (n = 4 blots; * p < 0.05). (H) Western blot analysis of LC3-I and II expression in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS. Western blot image quantification in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS for LC3-I in (I) and LC3-II in (J), normalized to ethanol control (Ethanol-LPS) (n = 4 blots; * p < 0.05). (K). Western blot analysis of LC3-I and II expression of Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS. Western blot image quantification of Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS for LC3-I in (L) and LC3-II in (M) normalized to ethanol control (Ethanol-LPS) (n = 3 blots; * p < 0.05).
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    FIGURE 7 The effect of acute ethanol-exposure on pAtg4B and microtubule associated protein 1 light chain-3B (LC3) I and II expression in macrophages. (A) WT-BMDM were exposed to vehicle or ethanol ± LPS. Atg4B was immunoprecipitated (IP) from whole-cell lysates of WT-BMDM using an anti-Atg4B antibody followed by IB analysis of <t>pSerine</t> and total Atg4B. IP with IgG control antibody was used as a negative control. (B) Western blot analysis of total Atg4B in the whole cell lysate used as input for the Atg4B IP. (C) SIRT2KO-BMDM were exposed to ethanol ± LPS. IP for Atg4B from whole-cell lysates of SIRT2KO-BMDM using an anti-Atg4B antibody followed by IB analysis of pSerine and total Atg4B. IP with IgG control antibody was used as a negative control. (D) Western blot analysis of total Atg4B in the whole cell lysate used as input for the Atg4B IP. (E) LC3-I and LC3-II expression were analyzed by western blot in WT-BMDM exposed to vehicle or ethanol ± LPS. Western blot image quantification of ethanol vs. vehicle-expose WT-BMDM± LPS, showing LC3-I in (F) and LC3-II in (G), normalized to vehicle control (Vehicle-LPS) (n = 4 blots; * p < 0.05). (H) Western blot analysis of LC3-I and II expression in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS. Western blot image quantification in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS for LC3-I in (I) and LC3-II in (J), normalized to ethanol control (Ethanol-LPS) (n = 4 blots; * p < 0.05). (K). Western blot analysis of LC3-I and II expression of Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS. Western blot image quantification of Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS for LC3-I in (L) and LC3-II in (M) normalized to ethanol control (Ethanol-LPS) (n = 3 blots; * p < 0.05).
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    FIGURE 7 The effect of acute ethanol-exposure on pAtg4B and microtubule associated protein 1 light chain-3B (LC3) I and II expression in macrophages. (A) WT-BMDM were exposed to vehicle or ethanol ± LPS. Atg4B was immunoprecipitated (IP) from whole-cell lysates of WT-BMDM using an anti-Atg4B antibody followed by IB analysis of <t>pSerine</t> and total Atg4B. IP with IgG control antibody was used as a negative control. (B) Western blot analysis of total Atg4B in the whole cell lysate used as input for the Atg4B IP. (C) SIRT2KO-BMDM were exposed to ethanol ± LPS. IP for Atg4B from whole-cell lysates of SIRT2KO-BMDM using an anti-Atg4B antibody followed by IB analysis of pSerine and total Atg4B. IP with IgG control antibody was used as a negative control. (D) Western blot analysis of total Atg4B in the whole cell lysate used as input for the Atg4B IP. (E) LC3-I and LC3-II expression were analyzed by western blot in WT-BMDM exposed to vehicle or ethanol ± LPS. Western blot image quantification of ethanol vs. vehicle-expose WT-BMDM± LPS, showing LC3-I in (F) and LC3-II in (G), normalized to vehicle control (Vehicle-LPS) (n = 4 blots; * p < 0.05). (H) Western blot analysis of LC3-I and II expression in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS. Western blot image quantification in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS for LC3-I in (I) and LC3-II in (J), normalized to ethanol control (Ethanol-LPS) (n = 4 blots; * p < 0.05). (K). Western blot analysis of LC3-I and II expression of Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS. Western blot image quantification of Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS for LC3-I in (L) and LC3-II in (M) normalized to ethanol control (Ethanol-LPS) (n = 3 blots; * p < 0.05).
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    Image Search Results


    FIGURE 7 The effect of acute ethanol-exposure on pAtg4B and microtubule associated protein 1 light chain-3B (LC3) I and II expression in macrophages. (A) WT-BMDM were exposed to vehicle or ethanol ± LPS. Atg4B was immunoprecipitated (IP) from whole-cell lysates of WT-BMDM using an anti-Atg4B antibody followed by IB analysis of pSerine and total Atg4B. IP with IgG control antibody was used as a negative control. (B) Western blot analysis of total Atg4B in the whole cell lysate used as input for the Atg4B IP. (C) SIRT2KO-BMDM were exposed to ethanol ± LPS. IP for Atg4B from whole-cell lysates of SIRT2KO-BMDM using an anti-Atg4B antibody followed by IB analysis of pSerine and total Atg4B. IP with IgG control antibody was used as a negative control. (D) Western blot analysis of total Atg4B in the whole cell lysate used as input for the Atg4B IP. (E) LC3-I and LC3-II expression were analyzed by western blot in WT-BMDM exposed to vehicle or ethanol ± LPS. Western blot image quantification of ethanol vs. vehicle-expose WT-BMDM± LPS, showing LC3-I in (F) and LC3-II in (G), normalized to vehicle control (Vehicle-LPS) (n = 4 blots; * p < 0.05). (H) Western blot analysis of LC3-I and II expression in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS. Western blot image quantification in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS for LC3-I in (I) and LC3-II in (J), normalized to ethanol control (Ethanol-LPS) (n = 4 blots; * p < 0.05). (K). Western blot analysis of LC3-I and II expression of Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS. Western blot image quantification of Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS for LC3-I in (L) and LC3-II in (M) normalized to ethanol control (Ethanol-LPS) (n = 3 blots; * p < 0.05).

    Journal: Frontiers in immunology

    Article Title: SIRT2-PFKP interaction dysregulates phagocytosis in macrophages with acute ethanol-exposure.

    doi: 10.3389/fimmu.2022.1079962

    Figure Lengend Snippet: FIGURE 7 The effect of acute ethanol-exposure on pAtg4B and microtubule associated protein 1 light chain-3B (LC3) I and II expression in macrophages. (A) WT-BMDM were exposed to vehicle or ethanol ± LPS. Atg4B was immunoprecipitated (IP) from whole-cell lysates of WT-BMDM using an anti-Atg4B antibody followed by IB analysis of pSerine and total Atg4B. IP with IgG control antibody was used as a negative control. (B) Western blot analysis of total Atg4B in the whole cell lysate used as input for the Atg4B IP. (C) SIRT2KO-BMDM were exposed to ethanol ± LPS. IP for Atg4B from whole-cell lysates of SIRT2KO-BMDM using an anti-Atg4B antibody followed by IB analysis of pSerine and total Atg4B. IP with IgG control antibody was used as a negative control. (D) Western blot analysis of total Atg4B in the whole cell lysate used as input for the Atg4B IP. (E) LC3-I and LC3-II expression were analyzed by western blot in WT-BMDM exposed to vehicle or ethanol ± LPS. Western blot image quantification of ethanol vs. vehicle-expose WT-BMDM± LPS, showing LC3-I in (F) and LC3-II in (G), normalized to vehicle control (Vehicle-LPS) (n = 4 blots; * p < 0.05). (H) Western blot analysis of LC3-I and II expression in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS. Western blot image quantification in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS for LC3-I in (I) and LC3-II in (J), normalized to ethanol control (Ethanol-LPS) (n = 4 blots; * p < 0.05). (K). Western blot analysis of LC3-I and II expression of Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS. Western blot image quantification of Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS for LC3-I in (L) and LC3-II in (M) normalized to ethanol control (Ethanol-LPS) (n = 3 blots; * p < 0.05).

    Article Snippet: Antibodies and reagents used for western blot and immunocytochemistry PFKP (Cell signaling Technology, Danvers, MA, CAT# 8164S), LC3 (Cell signaling Technology, Danvers, MA, CAT# E5Q2K: western blot), LC3 (Novus Biologicals, Centennial, Colorado, CAT# NC100-2220: immunocytochemistry), SIRT2 (Cell signaling Technology, Danvers, MA, CAT# D4050), Atg4B (Cell signaling Technology, Danvers, MA, CAT# 13507S), Rubicon (Cell signaling Technology, Danvers, MA, CAT# 8465S), Beclin-1 (Cell signaling Technology, CAT# 3495S), PFKM (Abcam, Waltham, Boston, USA,CAT#ab154804), VPS34 (Abcam, Waltham, Boston, CAT# ab124905), PFKL (Santa Cruz Biotechnology, Dallas, Texas, USA, CAT# sc393713), F4/80 (Invitrogen, Rockford, Illinois, USA, CAT# MA1-91124), Anti-rabbit Alexa Fluor 488 (Invitrogen, Rockford, Illinois, USA, CAT# A21206), Anti-rat Alexa Fluor 594 (Invitrogen, Rockford, Illinois, USA, CAT# A21209), Antirabbit Alexa Fluor 647 (Invitrogen, Rockford, Illinois, USA, CAT# A21245), Anti rabbit IgG control (Cell signaling Technology, Danvers, MA, CAT# 2729S), anti-mouse IgG, HRP-linked antibody (Cell signaling Technology, Danvers, MA, CAT# 7076), Anti-rabbit IgG, HRP-linked antibody (Cell signaling Technology, CAT# 7074), Acetyl lysine (Novus Biologicals, Centennial, CO, USA, CAT# NB 100-74339) pSerine (Origene, Rockville, MD, USA, CAT# AM00114PUN), Anti- Turbo GFP (tGFP) antibody (Origene, Rockville, MD, CAT# TA150041), DDK antibody (Origene, Rockville, MD, CAT# TA50011-100), Anti-Ubiquitin antibody (EMD Millipore, Burlington, Massachusetts, USA, CAT#5-944), Biotinylated anti-SIRT2 antibody (R&D system, Minneapolis, MN, USA, CAT# BAF4358), CPA (Novus Biologicals, CAT# NBP1-30993), Beta-actin(Abcam, Waltham, Boston, CAT# ab8226), ECL (Bio-Rad, Hercules, CA, USA, CAT# 1705061).

    Techniques: Expressing, Immunoprecipitation, Control, Negative Control, Western Blot