Journal: Scientific Reports

Article Title: Imaging the antimicrobial mechanism(s) of cathelicidin-2

doi: 10.1038/srep32948

Figure Lengend Snippet: E. coli 506 was co-incubated with different CATH-2 concentrations at 37 °C for different time points (1, 5, 10, 30, 60, 120 and 180 min). Each mixture was aliquoted at the various time intervals, serially diluted and spread plated on TSA plates. After 16 h at 37 °C plates were counted for surviving bacteria. Data represent three independent measurements (means ± SEM).

Article Snippet: MIC values of CATH-2 against E. coli ATCC 25922 (smooth LPS) were 2–8 fold higher than rough LPS containing E. coli K12 strains, suggesting that CATH-2 binds to the O-antigen part of LPS ( ).

Techniques: Incubation, Bacteria

Journal: Scientific Reports

Article Title: Imaging the antimicrobial mechanism(s) of cathelicidin-2

doi: 10.1038/srep32948

Figure Lengend Snippet: Various time lapses within a 6 min period are shown ( a ). Additionally, the fluorescent intensity of FITC-CATH-2 (0.9 μM) and PI (5.1 μM) was measured in both parts of a dividing bacterium ( E. coli 506), based on transverse linescans (yellow line) ( b ). Heat intensity plots show the specific binding sites of FITC-CATH-2 and PI ( c ). Bars, 1 μm.

Article Snippet: MIC values of CATH-2 against E. coli ATCC 25922 (smooth LPS) were 2–8 fold higher than rough LPS containing E. coli K12 strains, suggesting that CATH-2 binds to the O-antigen part of LPS ( ).

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: Imaging the antimicrobial mechanism(s) of cathelicidin-2

doi: 10.1038/srep32948

Figure Lengend Snippet: MIC values of CATH-2 against E. coli strains with rough and smooth LPS.

Article Snippet: MIC values of CATH-2 against E. coli ATCC 25922 (smooth LPS) were 2–8 fold higher than rough LPS containing E. coli K12 strains, suggesting that CATH-2 binds to the O-antigen part of LPS ( ).

Techniques:

Journal: Structure (London, England : 1993)

Article Title: Crystal Structure of β-Arrestin 2 in Complex with CXCR7 Phosphopeptide

doi: 10.1016/j.str.2020.06.002

Figure Lengend Snippet: (A) Overall structural snapshot of C7pp-bound βarr2 highlighting the loop regions. The C7pp peptide is shown as green sticks and the various loops in βarr2, i.e., the finger, middle, lariat, and C loops in the central crest, are colored in blue, black, orange, and olive, respectively. See also Figure S2.

Article Snippet: table ft1 table-wrap mode="anchored" t5 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies HRP-coupled Protein-L antibody GenScript Cat# M00098 Fab 30 This study N/A HRP-coupled M2 anti-FLAG antibody Sigma-Aldrich Cat# A8592; RRID: AB_439702 Bacterial and Virus Strains E. coli cells BL21(DE3)pLysS Novogen Cat# 71403 Chemicals, Peptides, and Recombinant Proteins Protein-L beads GE Healthcare Cat# 17547802 C7pp peptide NovoPep N/A LB media LPS solution Cat# LB-005 Chloramphenicol Bio Basic Inc. Cat# 56757 Kanamycin LPS solution Cat# KAN025 M9 minimal salt media Sigma-Aldrich Cat# MKCF5138 IPTG LPS solution Cat# IPTG025 PMSF Bio Basic Inc. Cat# PB0425 ParatoneⓇ N oil Sigma-Aldrich Cat# HR2-643 n -dodecyl-β-D-maltopyranoside Anatrace Cat# D310S Deposited Data Rat βarr2-C7pp complex structure This study 6K3F Bos Taurus βarr2 Zhan et al., 2011 3P2D Bos Taurus βarr2 Chen et al., 2017 5TV1 Bovine βarr1 Milano et al., 2002 1JSY Bos Taurus βarr1 Kang et al., 2009 3GC3 Rat βarr1 Shukla et al., 2013 4JQI Bos Taurus βarr1 Han et al., 2001 1G4M Ambystoma tigrinum visual arr1 Sutton et al., 2005 1SUJ Bos Taurus visual arr1 Hirsch et al., 1999 1CF1 Bos Taurus visual arr1 Granzin et al., 2012 3UGX Bos Taurus visual arr1 Granzin et al., 2012 3UGU Bos Taurus visual arr1 Granzin et al., 2015 4ZRG Bos Taurus visual arr1 Granzin et al., 1998 1AYR Mouse visual arr1 Kang et al., 2015 4ZWJ Mouse visual arr1 Zhou et al., 2017 5W0P Squid visual arr1 Bandyopadhyay et al., 2018 6BK9 Experimental Models: Cell Lines HEK-293 cells ATCC Cat# CRL-3216 HTLA cells Barnea et al., 2008 N/A Recombinant DNA pET28a-βarr2 1-410 This study N/A pET28a-βarr2 1-356 This study N/A Software and Algorithms GraphPad Prism 6.0 GraphPad graphpad.com PyMol 1.8 Schrodinger LLC pymol.org Phaser McCoy et al., 2007 phenix-online.org COOT Emsley and Cowtan, 2004 mrc-lmb.cam.ac.uk REFMAC5 Murshudov et al., 1997 ccp4.ac.uk/html/refmac5.html XDS Kabsch, 2010 xds.mpimf-heidelberg.mpg.de ProteinLynx Global Server (PLGS) 2.4 Waters waters.com DynamX 2.0 Waters waters.com Other Ni-sepharose affinity column GE Healthcare Cat# 17256801 Desalting column GE Healthcare Cat# 17085101 HiTrap Q sepharose column GE Healthcare Cat# 17115401 HiLoad 16/60 Superdex 200 GE Healthcare Cat# 28989335 HiTrap heparin column GE Healthcare Cat# 17040701 96-well MaxiSorp polystyrene plates Sigma-Aldrich Cat# P6366 Open in a separate window Key Resources Table Resource Availability Lead Contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Hyung Ho Lee ( rk.ca.uns@eelohgnuyh ).

Techniques:

Journal: Structure (London, England : 1993)

Article Title: Crystal Structure of β-Arrestin 2 in Complex with CXCR7 Phosphopeptide

doi: 10.1016/j.str.2020.06.002

Figure Lengend Snippet: Statistics for Data Collection and Refinement

Article Snippet: table ft1 table-wrap mode="anchored" t5 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies HRP-coupled Protein-L antibody GenScript Cat# M00098 Fab 30 This study N/A HRP-coupled M2 anti-FLAG antibody Sigma-Aldrich Cat# A8592; RRID: AB_439702 Bacterial and Virus Strains E. coli cells BL21(DE3)pLysS Novogen Cat# 71403 Chemicals, Peptides, and Recombinant Proteins Protein-L beads GE Healthcare Cat# 17547802 C7pp peptide NovoPep N/A LB media LPS solution Cat# LB-005 Chloramphenicol Bio Basic Inc. Cat# 56757 Kanamycin LPS solution Cat# KAN025 M9 minimal salt media Sigma-Aldrich Cat# MKCF5138 IPTG LPS solution Cat# IPTG025 PMSF Bio Basic Inc. Cat# PB0425 ParatoneⓇ N oil Sigma-Aldrich Cat# HR2-643 n -dodecyl-β-D-maltopyranoside Anatrace Cat# D310S Deposited Data Rat βarr2-C7pp complex structure This study 6K3F Bos Taurus βarr2 Zhan et al., 2011 3P2D Bos Taurus βarr2 Chen et al., 2017 5TV1 Bovine βarr1 Milano et al., 2002 1JSY Bos Taurus βarr1 Kang et al., 2009 3GC3 Rat βarr1 Shukla et al., 2013 4JQI Bos Taurus βarr1 Han et al., 2001 1G4M Ambystoma tigrinum visual arr1 Sutton et al., 2005 1SUJ Bos Taurus visual arr1 Hirsch et al., 1999 1CF1 Bos Taurus visual arr1 Granzin et al., 2012 3UGX Bos Taurus visual arr1 Granzin et al., 2012 3UGU Bos Taurus visual arr1 Granzin et al., 2015 4ZRG Bos Taurus visual arr1 Granzin et al., 1998 1AYR Mouse visual arr1 Kang et al., 2015 4ZWJ Mouse visual arr1 Zhou et al., 2017 5W0P Squid visual arr1 Bandyopadhyay et al., 2018 6BK9 Experimental Models: Cell Lines HEK-293 cells ATCC Cat# CRL-3216 HTLA cells Barnea et al., 2008 N/A Recombinant DNA pET28a-βarr2 1-410 This study N/A pET28a-βarr2 1-356 This study N/A Software and Algorithms GraphPad Prism 6.0 GraphPad graphpad.com PyMol 1.8 Schrodinger LLC pymol.org Phaser McCoy et al., 2007 phenix-online.org COOT Emsley and Cowtan, 2004 mrc-lmb.cam.ac.uk REFMAC5 Murshudov et al., 1997 ccp4.ac.uk/html/refmac5.html XDS Kabsch, 2010 xds.mpimf-heidelberg.mpg.de ProteinLynx Global Server (PLGS) 2.4 Waters waters.com DynamX 2.0 Waters waters.com Other Ni-sepharose affinity column GE Healthcare Cat# 17256801 Desalting column GE Healthcare Cat# 17085101 HiTrap Q sepharose column GE Healthcare Cat# 17115401 HiLoad 16/60 Superdex 200 GE Healthcare Cat# 28989335 HiTrap heparin column GE Healthcare Cat# 17040701 96-well MaxiSorp polystyrene plates Sigma-Aldrich Cat# P6366 Open in a separate window Key Resources Table Resource Availability Lead Contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Hyung Ho Lee ( rk.ca.uns@eelohgnuyh ).

Techniques:

Journal: Structure (London, England : 1993)

Article Title: Crystal Structure of β-Arrestin 2 in Complex with CXCR7 Phosphopeptide

doi: 10.1016/j.str.2020.06.002

Figure Lengend Snippet: (A) Structural comparisons of the finger, middle, lariat, and C loops in C7pp-bound βarr2 (PDB: 6K3F, blue), R175E visual arr1 (PDB: 4ZRG, orange), IP6-bound βarr2 (PDB: 5TV1, light blue), and V2Rpp-bound βarr1 (PDB: 4JQI, light cyan).

Article Snippet: table ft1 table-wrap mode="anchored" t5 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies HRP-coupled Protein-L antibody GenScript Cat# M00098 Fab 30 This study N/A HRP-coupled M2 anti-FLAG antibody Sigma-Aldrich Cat# A8592; RRID: AB_439702 Bacterial and Virus Strains E. coli cells BL21(DE3)pLysS Novogen Cat# 71403 Chemicals, Peptides, and Recombinant Proteins Protein-L beads GE Healthcare Cat# 17547802 C7pp peptide NovoPep N/A LB media LPS solution Cat# LB-005 Chloramphenicol Bio Basic Inc. Cat# 56757 Kanamycin LPS solution Cat# KAN025 M9 minimal salt media Sigma-Aldrich Cat# MKCF5138 IPTG LPS solution Cat# IPTG025 PMSF Bio Basic Inc. Cat# PB0425 ParatoneⓇ N oil Sigma-Aldrich Cat# HR2-643 n -dodecyl-β-D-maltopyranoside Anatrace Cat# D310S Deposited Data Rat βarr2-C7pp complex structure This study 6K3F Bos Taurus βarr2 Zhan et al., 2011 3P2D Bos Taurus βarr2 Chen et al., 2017 5TV1 Bovine βarr1 Milano et al., 2002 1JSY Bos Taurus βarr1 Kang et al., 2009 3GC3 Rat βarr1 Shukla et al., 2013 4JQI Bos Taurus βarr1 Han et al., 2001 1G4M Ambystoma tigrinum visual arr1 Sutton et al., 2005 1SUJ Bos Taurus visual arr1 Hirsch et al., 1999 1CF1 Bos Taurus visual arr1 Granzin et al., 2012 3UGX Bos Taurus visual arr1 Granzin et al., 2012 3UGU Bos Taurus visual arr1 Granzin et al., 2015 4ZRG Bos Taurus visual arr1 Granzin et al., 1998 1AYR Mouse visual arr1 Kang et al., 2015 4ZWJ Mouse visual arr1 Zhou et al., 2017 5W0P Squid visual arr1 Bandyopadhyay et al., 2018 6BK9 Experimental Models: Cell Lines HEK-293 cells ATCC Cat# CRL-3216 HTLA cells Barnea et al., 2008 N/A Recombinant DNA pET28a-βarr2 1-410 This study N/A pET28a-βarr2 1-356 This study N/A Software and Algorithms GraphPad Prism 6.0 GraphPad graphpad.com PyMol 1.8 Schrodinger LLC pymol.org Phaser McCoy et al., 2007 phenix-online.org COOT Emsley and Cowtan, 2004 mrc-lmb.cam.ac.uk REFMAC5 Murshudov et al., 1997 ccp4.ac.uk/html/refmac5.html XDS Kabsch, 2010 xds.mpimf-heidelberg.mpg.de ProteinLynx Global Server (PLGS) 2.4 Waters waters.com DynamX 2.0 Waters waters.com Other Ni-sepharose affinity column GE Healthcare Cat# 17256801 Desalting column GE Healthcare Cat# 17085101 HiTrap Q sepharose column GE Healthcare Cat# 17115401 HiLoad 16/60 Superdex 200 GE Healthcare Cat# 28989335 HiTrap heparin column GE Healthcare Cat# 17040701 96-well MaxiSorp polystyrene plates Sigma-Aldrich Cat# P6366 Open in a separate window Key Resources Table Resource Availability Lead Contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Hyung Ho Lee ( rk.ca.uns@eelohgnuyh ).

Techniques:

Journal: Structure (London, England : 1993)

Article Title: Crystal Structure of β-Arrestin 2 in Complex with CXCR7 Phosphopeptide

doi: 10.1016/j.str.2020.06.002

Figure Lengend Snippet: (A) An overall distinct binding mode of C7pp with βarr2 (PDB: 6K3F, green) compared with the V2Rpp-βarr1 complex (PDB: 4JQI, light cyan). The N domains from the crystal structures of the C7pp-βarr2 complex and V2Rpp-βarr1 are superimposed and the respective phosphopeptides are highlighted for comparison.

Article Snippet: table ft1 table-wrap mode="anchored" t5 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies HRP-coupled Protein-L antibody GenScript Cat# M00098 Fab 30 This study N/A HRP-coupled M2 anti-FLAG antibody Sigma-Aldrich Cat# A8592; RRID: AB_439702 Bacterial and Virus Strains E. coli cells BL21(DE3)pLysS Novogen Cat# 71403 Chemicals, Peptides, and Recombinant Proteins Protein-L beads GE Healthcare Cat# 17547802 C7pp peptide NovoPep N/A LB media LPS solution Cat# LB-005 Chloramphenicol Bio Basic Inc. Cat# 56757 Kanamycin LPS solution Cat# KAN025 M9 minimal salt media Sigma-Aldrich Cat# MKCF5138 IPTG LPS solution Cat# IPTG025 PMSF Bio Basic Inc. Cat# PB0425 ParatoneⓇ N oil Sigma-Aldrich Cat# HR2-643 n -dodecyl-β-D-maltopyranoside Anatrace Cat# D310S Deposited Data Rat βarr2-C7pp complex structure This study 6K3F Bos Taurus βarr2 Zhan et al., 2011 3P2D Bos Taurus βarr2 Chen et al., 2017 5TV1 Bovine βarr1 Milano et al., 2002 1JSY Bos Taurus βarr1 Kang et al., 2009 3GC3 Rat βarr1 Shukla et al., 2013 4JQI Bos Taurus βarr1 Han et al., 2001 1G4M Ambystoma tigrinum visual arr1 Sutton et al., 2005 1SUJ Bos Taurus visual arr1 Hirsch et al., 1999 1CF1 Bos Taurus visual arr1 Granzin et al., 2012 3UGX Bos Taurus visual arr1 Granzin et al., 2012 3UGU Bos Taurus visual arr1 Granzin et al., 2015 4ZRG Bos Taurus visual arr1 Granzin et al., 1998 1AYR Mouse visual arr1 Kang et al., 2015 4ZWJ Mouse visual arr1 Zhou et al., 2017 5W0P Squid visual arr1 Bandyopadhyay et al., 2018 6BK9 Experimental Models: Cell Lines HEK-293 cells ATCC Cat# CRL-3216 HTLA cells Barnea et al., 2008 N/A Recombinant DNA pET28a-βarr2 1-410 This study N/A pET28a-βarr2 1-356 This study N/A Software and Algorithms GraphPad Prism 6.0 GraphPad graphpad.com PyMol 1.8 Schrodinger LLC pymol.org Phaser McCoy et al., 2007 phenix-online.org COOT Emsley and Cowtan, 2004 mrc-lmb.cam.ac.uk REFMAC5 Murshudov et al., 1997 ccp4.ac.uk/html/refmac5.html XDS Kabsch, 2010 xds.mpimf-heidelberg.mpg.de ProteinLynx Global Server (PLGS) 2.4 Waters waters.com DynamX 2.0 Waters waters.com Other Ni-sepharose affinity column GE Healthcare Cat# 17256801 Desalting column GE Healthcare Cat# 17085101 HiTrap Q sepharose column GE Healthcare Cat# 17115401 HiLoad 16/60 Superdex 200 GE Healthcare Cat# 28989335 HiTrap heparin column GE Healthcare Cat# 17040701 96-well MaxiSorp polystyrene plates Sigma-Aldrich Cat# P6366 Open in a separate window Key Resources Table Resource Availability Lead Contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Hyung Ho Lee ( rk.ca.uns@eelohgnuyh ).

Techniques: Binding Assay

Journal: Structure (London, England : 1993)

Article Title: Crystal Structure of β-Arrestin 2 in Complex with CXCR7 Phosphopeptide

doi: 10.1016/j.str.2020.06.002

Figure Lengend Snippet: Surface representation of the overall electrostatic potential of the C7pp-bound βarr2 structure. C7pp is shown as green sticks. In the positive electrostatic surface of the N domain, the four hotspots for C7pp binding are shown in the dotted rectangle or circles (F, P, A, and B). The panels on the right represent the detailed interactions at the βarr2-C7pp interface and specific interactions of the phosphates with various residues in βarr2. See also Figures S1B and S6.

Article Snippet: table ft1 table-wrap mode="anchored" t5 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies HRP-coupled Protein-L antibody GenScript Cat# M00098 Fab 30 This study N/A HRP-coupled M2 anti-FLAG antibody Sigma-Aldrich Cat# A8592; RRID: AB_439702 Bacterial and Virus Strains E. coli cells BL21(DE3)pLysS Novogen Cat# 71403 Chemicals, Peptides, and Recombinant Proteins Protein-L beads GE Healthcare Cat# 17547802 C7pp peptide NovoPep N/A LB media LPS solution Cat# LB-005 Chloramphenicol Bio Basic Inc. Cat# 56757 Kanamycin LPS solution Cat# KAN025 M9 minimal salt media Sigma-Aldrich Cat# MKCF5138 IPTG LPS solution Cat# IPTG025 PMSF Bio Basic Inc. Cat# PB0425 ParatoneⓇ N oil Sigma-Aldrich Cat# HR2-643 n -dodecyl-β-D-maltopyranoside Anatrace Cat# D310S Deposited Data Rat βarr2-C7pp complex structure This study 6K3F Bos Taurus βarr2 Zhan et al., 2011 3P2D Bos Taurus βarr2 Chen et al., 2017 5TV1 Bovine βarr1 Milano et al., 2002 1JSY Bos Taurus βarr1 Kang et al., 2009 3GC3 Rat βarr1 Shukla et al., 2013 4JQI Bos Taurus βarr1 Han et al., 2001 1G4M Ambystoma tigrinum visual arr1 Sutton et al., 2005 1SUJ Bos Taurus visual arr1 Hirsch et al., 1999 1CF1 Bos Taurus visual arr1 Granzin et al., 2012 3UGX Bos Taurus visual arr1 Granzin et al., 2012 3UGU Bos Taurus visual arr1 Granzin et al., 2015 4ZRG Bos Taurus visual arr1 Granzin et al., 1998 1AYR Mouse visual arr1 Kang et al., 2015 4ZWJ Mouse visual arr1 Zhou et al., 2017 5W0P Squid visual arr1 Bandyopadhyay et al., 2018 6BK9 Experimental Models: Cell Lines HEK-293 cells ATCC Cat# CRL-3216 HTLA cells Barnea et al., 2008 N/A Recombinant DNA pET28a-βarr2 1-410 This study N/A pET28a-βarr2 1-356 This study N/A Software and Algorithms GraphPad Prism 6.0 GraphPad graphpad.com PyMol 1.8 Schrodinger LLC pymol.org Phaser McCoy et al., 2007 phenix-online.org COOT Emsley and Cowtan, 2004 mrc-lmb.cam.ac.uk REFMAC5 Murshudov et al., 1997 ccp4.ac.uk/html/refmac5.html XDS Kabsch, 2010 xds.mpimf-heidelberg.mpg.de ProteinLynx Global Server (PLGS) 2.4 Waters waters.com DynamX 2.0 Waters waters.com Other Ni-sepharose affinity column GE Healthcare Cat# 17256801 Desalting column GE Healthcare Cat# 17085101 HiTrap Q sepharose column GE Healthcare Cat# 17115401 HiLoad 16/60 Superdex 200 GE Healthcare Cat# 28989335 HiTrap heparin column GE Healthcare Cat# 17040701 96-well MaxiSorp polystyrene plates Sigma-Aldrich Cat# P6366 Open in a separate window Key Resources Table Resource Availability Lead Contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Hyung Ho Lee ( rk.ca.uns@eelohgnuyh ).

Techniques: Binding Assay

Journal: Structure (London, England : 1993)

Article Title: Crystal Structure of β-Arrestin 2 in Complex with CXCR7 Phosphopeptide

doi: 10.1016/j.str.2020.06.002

Figure Lengend Snippet: Key Resources Table

Article Snippet: table ft1 table-wrap mode="anchored" t5 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies HRP-coupled Protein-L antibody GenScript Cat# M00098 Fab 30 This study N/A HRP-coupled M2 anti-FLAG antibody Sigma-Aldrich Cat# A8592; RRID: AB_439702 Bacterial and Virus Strains E. coli cells BL21(DE3)pLysS Novogen Cat# 71403 Chemicals, Peptides, and Recombinant Proteins Protein-L beads GE Healthcare Cat# 17547802 C7pp peptide NovoPep N/A LB media LPS solution Cat# LB-005 Chloramphenicol Bio Basic Inc. Cat# 56757 Kanamycin LPS solution Cat# KAN025 M9 minimal salt media Sigma-Aldrich Cat# MKCF5138 IPTG LPS solution Cat# IPTG025 PMSF Bio Basic Inc. Cat# PB0425 ParatoneⓇ N oil Sigma-Aldrich Cat# HR2-643 n -dodecyl-β-D-maltopyranoside Anatrace Cat# D310S Deposited Data Rat βarr2-C7pp complex structure This study 6K3F Bos Taurus βarr2 Zhan et al., 2011 3P2D Bos Taurus βarr2 Chen et al., 2017 5TV1 Bovine βarr1 Milano et al., 2002 1JSY Bos Taurus βarr1 Kang et al., 2009 3GC3 Rat βarr1 Shukla et al., 2013 4JQI Bos Taurus βarr1 Han et al., 2001 1G4M Ambystoma tigrinum visual arr1 Sutton et al., 2005 1SUJ Bos Taurus visual arr1 Hirsch et al., 1999 1CF1 Bos Taurus visual arr1 Granzin et al., 2012 3UGX Bos Taurus visual arr1 Granzin et al., 2012 3UGU Bos Taurus visual arr1 Granzin et al., 2015 4ZRG Bos Taurus visual arr1 Granzin et al., 1998 1AYR Mouse visual arr1 Kang et al., 2015 4ZWJ Mouse visual arr1 Zhou et al., 2017 5W0P Squid visual arr1 Bandyopadhyay et al., 2018 6BK9 Experimental Models: Cell Lines HEK-293 cells ATCC Cat# CRL-3216 HTLA cells Barnea et al., 2008 N/A Recombinant DNA pET28a-βarr2 1-410 This study N/A pET28a-βarr2 1-356 This study N/A Software and Algorithms GraphPad Prism 6.0 GraphPad graphpad.com PyMol 1.8 Schrodinger LLC pymol.org Phaser McCoy et al., 2007 phenix-online.org COOT Emsley and Cowtan, 2004 mrc-lmb.cam.ac.uk REFMAC5 Murshudov et al., 1997 ccp4.ac.uk/html/refmac5.html XDS Kabsch, 2010 xds.mpimf-heidelberg.mpg.de ProteinLynx Global Server (PLGS) 2.4 Waters waters.com DynamX 2.0 Waters waters.com Other Ni-sepharose affinity column GE Healthcare Cat# 17256801 Desalting column GE Healthcare Cat# 17085101 HiTrap Q sepharose column GE Healthcare Cat# 17115401 HiLoad 16/60 Superdex 200 GE Healthcare Cat# 28989335 HiTrap heparin column GE Healthcare Cat# 17040701 96-well MaxiSorp polystyrene plates Sigma-Aldrich Cat# P6366 Open in a separate window Key Resources Table Resource Availability Lead Contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Hyung Ho Lee ( rk.ca.uns@eelohgnuyh ).

Techniques: Recombinant, Software, Affinity Column

Journal: Physiological Research

Article Title: Berberine Exerts Neuroprotective Effects in Alzheimer’s Disease by Switching Microglia M1/M2 Polarization Through PI3K-AKT Signaling

doi: 10.33549/physiolres.935410

Figure Lengend Snippet: ( A ) CCK8 assay was conducted to analyze the toxicity of different doses of BBR (1 μM, 2 μM, 4 μM, 8 μM, or 16 μM) in HMC3 cells. ( B-F ) HMC3 cells were divided into six groups: control, BBR (8 μM), LPS, LPS+BBR (2 μM), LPS+BBR (4 μM), and LPS+BBR (8 μM). For BBR and LPS co-treated group, HMC3 cells were treated with BBR for 3 h, followed by LPS exposure for 24 h. ( B and C ) IF assay was carried out to evaluate the levels of two indicators of inflammation, including iNOS and COX2. ( D-F ) ELISA was implemented to analyze the release of three pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) in the culture supernatant. ns: no statistically significant difference, * P< 0.05, ** P< 0.01, *** P< 0.001.

Article Snippet: LPS (Cat. No. L8880) and BBR (Cat. No. SB8130) were purchased from Solarbio life sciences (Beijing, China).

Techniques: CCK-8 Assay, Control, Enzyme-linked Immunosorbent Assay

Journal: Physiological Research

Article Title: Berberine Exerts Neuroprotective Effects in Alzheimer’s Disease by Switching Microglia M1/M2 Polarization Through PI3K-AKT Signaling

doi: 10.33549/physiolres.935410

Figure Lengend Snippet: ( A-D ) Aβ 1-42 -exposed SH-SY5Y cells were cultured in the conditioned medium from control, BBR-stimulated, LPS-stimulated, or BBR and LPS co-treated HMC3 cells. ( A ) CCK8 assay was employed to analyze the viability of SH-SY5Y cells. ( B and C ) The MMP and ROS in SH-SY5Y cells were determined through IF staining method of JC-1 and DCFH-DA, respectively. ( D ) Western blot assay was conducted to measure the protein levels of two apoptosis-associated markers (cleaved CASP3 and Bax) in SH-SY5Y cells. ns: no statistically significant difference, * P< 0.05, *** P< 0.001.

Article Snippet: LPS (Cat. No. L8880) and BBR (Cat. No. SB8130) were purchased from Solarbio life sciences (Beijing, China).

Techniques: Cell Culture, Control, CCK-8 Assay, Staining, Western Blot

Journal: Physiological Research

Article Title: Berberine Exerts Neuroprotective Effects in Alzheimer’s Disease by Switching Microglia M1/M2 Polarization Through PI3K-AKT Signaling

doi: 10.33549/physiolres.935410

Figure Lengend Snippet: ( A-C ) HMC3 cells were divided into five groups: control, BBR (8 μM), LPS, LPS+BBR (8 μM), LPS+BBR (8 μM) + LY294002. For BBR and LPS co-treated group, HMC3 cells were treated with BBR for 3 h, followed by LPS exposure for 24 h. For LPS, BBR, and LY294002 co-treated group, HMC3 cells were treated with BBR for 3 h, followed by LPS and LY294002 stimulation for 24 h. ( A ) The levels of p-PI3K, PI3K, p-AKT, and AKT in HMC3 cells were determined by western blot assay. ( B and C ) ELISA was employed to detect the release of two pro-inflammatory cytokines (IL-1β and IL-6) in the culture supernatant of HMC3 cells. ns: no statistically significant difference, ** P< 0.01, *** P< 0.001.

Article Snippet: LPS (Cat. No. L8880) and BBR (Cat. No. SB8130) were purchased from Solarbio life sciences (Beijing, China).

Techniques: Control, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Pharmacology

Article Title: Targeting IgG Autoantibodies for Improved Cytotoxicity of Bactericidal Permeability Increasing Protein in Cystic Fibrosis

doi: 10.3389/fphar.2020.01098

Figure Lengend Snippet: Elevated levels of BPI present in plasma and BAL of people with CF. (A , B) LPS levels in CF plasma and BAL correlated with CF-ABLE score. Quantification of an association between variables was achieved by Spearman correlation. (C , D) Comparative analysis of BPI present in plasma or BAL of Phe508del homozygous PWCF (CF), healthy controls (HC) or NCFB patients was performed by ELISA. (C) BPI levels were significantly increased in plasma of CF compared to HC (n=22 and n=14 subjects per group, respectively, p=0.007, Mann Whitney U-test). (D) BAL levels of BPI were significantly increased in CF (n=5) compared to HC (n=4) or NCFB patients (n=5) (p<0.0001, One-way ANOVA, followed by Bonferroni post-hoc test for selected groups). (E) BAL samples from HC, CF, NCFB or COPD were subjected to SDS-PAGE and Western blot analysis for BPI. An immuno-band of increased intensity for BPI was detected in CF BAL samples (top panels). Lower panel, a control immunoblot to ensure BPI specificity. The blot was halved, with one half probed with secondary antibody only with no BPI immune-bands visible (↑ indicates where blot was cut). Human BPI (hBPI) was used as a positive control. All measurements are means ± SEM from biological replicates.

Article Snippet: Lipopolysaccharide (LPS) was quantified in plasma and BAL of PWCF ( ) using an LPS ELISA (Cusabio: catalog number CSB-E09945h).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, SDS Page, Western Blot, Control, Positive Control

Journal: Frontiers in Pharmacology

Article Title: Targeting IgG Autoantibodies for Improved Cytotoxicity of Bactericidal Permeability Increasing Protein in Cystic Fibrosis

doi: 10.3389/fphar.2020.01098

Figure Lengend Snippet: Increased levels of plasma autoantibodies and IgG-bound BPI in CF airway samples. (A) IgG autoantibodies against BPI were quantified in plasma of PWCF (CF, n=30) or healthy controls (HC, n=37). A significant increase in the titre of BPI autoantibodies were detected in CF (p<0.0001, Mann-Whitney U test). Positivity was set as 10 U/ml as indicated by the hatched line. All CF samples were positive for BPI autoantibodies. Increased circulating IgG BPI antibody level in CF individuals determined by ELISA. (B) Plasma samples from HC (n=38), CF (n=28) and CF individuals receiving ivacaftor treatment (n=10). No difference in levels of circulating anti-BPI IgG autoantibodies between CF and CF individuals receiving ivacaftor treatment (p=0.39). (C) Representative Coomassie blue stained SDS gel of purified IgG from CF BAL. Protein G Sepharose was used to isolate IgG from BAL of Phe508del homozygous PWCF. Starting BAL sample (St), unbound material (Un) or purified bound IgG (Bd) with or without DTT reduction are presented. Purified IgG (150 kDa, closed arrow), and the heavy (50 kDa) and light chains (25 kDa) of IgG are indicated (open arrows) (1 representative images of n = 5 biological repeats). (D) Protein G Sepharose was used to isolate IgG-BPI complexes from CF BAL. Reactions were analyzed by immunoblotting for BPI positivity using a mouse monoclonal anti-BPI antibody. Starting BAL sample (St), unbound material (Un) or IgG-bound BPI (Bd) are presented. Human BPI (hBPI) was used as a positive control (55 kDa). A control immunoblot to ensure BPI specificity omitted primary antibody (right hand panel), with no immune-band apparent. 3 representative images of n=5 biological repeats.

Article Snippet: Lipopolysaccharide (LPS) was quantified in plasma and BAL of PWCF ( ) using an LPS ELISA (Cusabio: catalog number CSB-E09945h).

Techniques: Clinical Proteomics, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay, Staining, SDS-Gel, Purification, Western Blot, Positive Control, Control