puno tlr2 plus puno tlr1  (InvivoGen)

Journal: Scientific Reports

Article Title: Peptidoglycan mediates Leptospira outer membrane protein Loa22 to toll-like receptor 2 for inflammatory interaction: a novel innate immune recognition

doi: 10.1038/s41598-020-79662-8

Figure Lengend Snippet: Characterization of TLR2 binding proteins from Leptospira . ( A ) Immuno-precipitant of purified TLR2 protein and Loa22 protein. Anti-TLR2, anti-LipL32 and anti-Loa22 antibodies were used to detect the presence of these proteins. Lane 1: anti-V5 antibody activated Protein A beads incubated with HEK293-TLR2 cell lysate; lane 2: anti-V5 antibody activated Protein A beads incubated with LOMP; lane 3: LOMP; lane 4: Co-IP of TLR2 and LOMP fractions. ( B ) The expression of Loa22 protein in different species and fractions of Leptospira . Recombinant Loa22 (rLoa22) was used as positive control (lane 1). Peptidoglycan from L. santarosai (LPGN) was also used to detect the presence of Loa22 protein (lane 2). LOMPs from different species of Leptospires were used to detect the presence or absence of Loa22 protein including pathogenic L. interrogans (lane 3), non-pathogenic L. biflexa (lane 4), and L. santarosai (lane 5). Rabbit polyclone anti-Loa22 antibody was use to recognize the protein. ( C ) Purification of the Loa22 and mutation variants. Wild type and mutation variants were analyzed by SDS-PAGE (lower panel) and Western blot (upper panel; recognized by anti 6XHis tag antibody) Lane 1: WT Loa222; lane 2; Loa22D122A; lane 3: Loa22R143A. ( D ) Size exclusion chromatography of purified Loa22 protein. The single peak of purified Loa22 protein with molecular mass about 22 kDa indicated that the protein was of uniform conformation. The standard markers were ferritin (440 kDa), β-galactosidase (125 kDa), albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and ribonucleaseA (15 kDa).

Article Snippet: The human TLR2 gene was sub-cloned from plasmid pUNO-TLR2 (Invivogen, San Diego, CA) and inserted into a lentivirus expression vector with a V5 tag at the C-terminus.

Techniques: Binding Assay, Purification, Incubation, Co-Immunoprecipitation Assay, Expressing, Recombinant, Positive Control, Mutagenesis, SDS Page, Western Blot, Size-exclusion Chromatography

Journal: Scientific Reports

Article Title: Peptidoglycan mediates Leptospira outer membrane protein Loa22 to toll-like receptor 2 for inflammatory interaction: a novel innate immune recognition

doi: 10.1038/s41598-020-79662-8

Figure Lengend Snippet: Loa22 co-localized with TLR2 on HEK293-TLR2 cells. HEK293-TLR2 cell were cultured to 70% confluence and serum free for 16 h before adding the stimulation agents (0.1 μg/ml). The cells were incubated with the stimulation agents for 4 h and then fixed to against with the anti-V5 antibody (1:5000), anti-Loa22 antibody (1:10,000). The relative Alex488 and Alex594 conjugated secondary antibodies were used to stained TLR2 and Loa22 proteins, respectively. ( A ) rLoa22WT was incubated with HEK293 cell. ( B ) rLoa22WT-LPGN was incubated with HEK293-TLR2 cell. ( C ) rLoa22D122A-LPGN was incubated with HEK293-TLR2 cell. ( D ) rLoa22R143A-LPGN was incubated with HEK293-TLR2 cell. ( E ) Pam 3 CSK 4 Rhodamine was incubated with HEK293-TLR2 cell. The nucleus was stained by DAPI (blue) and TLR2 was stained by Alexa488 (green). Loa22 was stained by Alexa594 (red). The yellow color indicated that the two proteins were co-localized in HEK293-TLR2 cell.

Article Snippet: The human TLR2 gene was sub-cloned from plasmid pUNO-TLR2 (Invivogen, San Diego, CA) and inserted into a lentivirus expression vector with a V5 tag at the C-terminus.

Techniques: Cell Culture, Incubation, Staining

Journal: Scientific Reports

Article Title: Peptidoglycan mediates Leptospira outer membrane protein Loa22 to toll-like receptor 2 for inflammatory interaction: a novel innate immune recognition

doi: 10.1038/s41598-020-79662-8

Figure Lengend Snippet: In vitro assay of the interaction between TLR2 and rLoa22-LPGN complexes. ( A ) ELISA assay of the interaction between TLR2 and rLoa22. ( B ) AFM force-distance curves of the interaction between TLR2 and rLoa22. ( C ) The binding frequency of the interaction between TLR2 and rLoa22. TLR2 interacted to rLoa22WT and mutation variants, rLoa22D122A and rLoa22R143A in the presence or absence of LPGN. BSA was used as negative control. p < 0.05; p < 0.01.

Article Snippet: The human TLR2 gene was sub-cloned from plasmid pUNO-TLR2 (Invivogen, San Diego, CA) and inserted into a lentivirus expression vector with a V5 tag at the C-terminus.

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Binding Assay, Mutagenesis, Negative Control

Journal: Scientific Reports

Article Title: Peptidoglycan mediates Leptospira outer membrane protein Loa22 to toll-like receptor 2 for inflammatory interaction: a novel innate immune recognition

doi: 10.1038/s41598-020-79662-8

Figure Lengend Snippet: TLR2 downstream signaling cascades assays after rLoa22-LPGN complexes stimulation. HEK293-TLR2 cells were cultured to 70% confluence and serum free for 16 h before adding the stimulation agents (0.1 μg/ml). The cells were incubated with the stimulation agents for 1 h and collected for western blot analysis. ( A ) rLoa22-LPGN complexes stimulated HEK293 and HEK293-TLR2 cells for 1 h and downstream signaling cascades were assayed. ( B ) rLoa22-LPGN complexes stimulated HK2 cells in the presence and absence of TLR2 antibody and downstream signaling cascades were assayed. Anti-TLR2 antibody (1 μg/ml) was used to pretreated HK2 cell for 1 h before adding the rLoa22-LPGN complexes. ( C ) Luciferase assay of NF-κB in HEK293 cell. HEK293-TLR2 cell transfected with pNF-κB luc plasmid and NF-κB translocation activity was assayed by luciferase reporter assay. PBS was used as control and TNF-α was used as positive control. rLipL32 and rLoa22-LPGN were used to stimulate TLR2 downstream signaling cascades and promoted NF-κB translocated into nuclus. p < 0.05; p < 0.01.

Article Snippet: The human TLR2 gene was sub-cloned from plasmid pUNO-TLR2 (Invivogen, San Diego, CA) and inserted into a lentivirus expression vector with a V5 tag at the C-terminus.

Techniques: Cell Culture, Incubation, Western Blot, Luciferase, Transfection, Plasmid Preparation, Translocation Assay, Activity Assay, Reporter Assay, Positive Control

Journal: Scientific Reports

Article Title: Peptidoglycan mediates Leptospira outer membrane protein Loa22 to toll-like receptor 2 for inflammatory interaction: a novel innate immune recognition

doi: 10.1038/s41598-020-79662-8

Figure Lengend Snippet: Inflammatory responses induced by rLoa22-LPGN in HEK293-TLR2 cells. HEK293-TLR2 cells were cultured to 70% confluence and changed to serum free conditions for 16 h. The stimulation agents (0.1 μg/ml) were added to stimulate the downstream inflammatory responses from TLR2 signaling such as CXCL8/IL8 , hCCL2/MCP-1 , and hTNF-α . ( A ) Stimulation of the expression of CXCL8/IL8 mRNA. ( B ) Stimulation of the expression of CXCL8/IL8 protein. ( C ) Stimulation of the expression of hCCL2/MCP-1 mRNA. ( D ) Stimulation of the expression of hCCL2/MCP-1 protein ( E ) Stimulation of the expression of hTNF-α mRNA. ( F ) Stimulation of the expression of hTNF-α protein. Inflammatory responses stimulated by Loa22 and its variants (rLoa22WT, rLoa22D122A, and rLoa22R143A) in the presence or absence of LPGN. The results of mRNA levels in different genes are displayed as the transcript levels of the analyzed genes relative to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) transcript level. The secretion cytokines were measured by ELISA. p < 0.05; p < 0.01; p < 0.001.

Article Snippet: The human TLR2 gene was sub-cloned from plasmid pUNO-TLR2 (Invivogen, San Diego, CA) and inserted into a lentivirus expression vector with a V5 tag at the C-terminus.

Techniques: Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Peptidoglycan mediates Leptospira outer membrane protein Loa22 to toll-like receptor 2 for inflammatory interaction: a novel innate immune recognition

doi: 10.1038/s41598-020-79662-8

Figure Lengend Snippet: Inflammatory responses induced by rLoa22-LPGN in transient transfection HEK293 cells. Several plasmids (TLR2-TLR1, TLR2-TLR1, and TLR2 only) were transiently transfected into HEK-293 cell and the inflammatory responses induced by rLoa22-LPGN complex were assayed including hCXCL8/IL8, hCCL2/MCP-1, and hTNF-α. ( A ) Stimulation of the expression of CXCL8/IL8 mRNA. ( B ) Stimulation of the expression of CXCL8/IL8 protein. ( C ) Stimulation of the expression of hCCL2/MCP-1 mRNA. ( D ) Stimulation of the expression of hCCL2/MCP-1 protein. ( E ) Stimulation of the expression of hTNF-α mRNA. (F) Stimulation of the expression of hTNF-α protein. HEK-293-pUNO was used as mock control. p < 0.05; p < 0.01.

Article Snippet: The human TLR2 gene was sub-cloned from plasmid pUNO-TLR2 (Invivogen, San Diego, CA) and inserted into a lentivirus expression vector with a V5 tag at the C-terminus.

Techniques: Transfection, Expressing

Journal: bioRxiv

Article Title: Peptidoglycan Mediates Loa22 and Toll-like Receptor 2 Interactions in Pathogenic Leptospira

doi: 10.1101/520288

Figure Lengend Snippet: Characterization and bioinformatic analysis of Loa22 protein. (A) Domains prediction of Loa22. Loa22 contains 195 amino acids and the domains preditions revealed that the N terminal contains signal peptide ( – ) and C terminal of contains OmpA_C like domain (78-186). (B) Immuno-precipitant of purified TLR2 protein and Loa22 protein. Anti-TLR2, anti-LipL32 and anti-Loa22 antibodies were used to detect the presence of these proteins. (C) Sequence alignment of Loa22. Loa22 sequence was aligned with other OmpA domain protein including ABOmpA and Pal protein from A. baumannii and E. coli, respectively. Two pivotal residues (Asp122 and Arg143; red arrows) were highly conserved in Loa22 and other OmpA domain proteins and the two residues were responsible for peptidylglycan (PGN) binding. (D) Purification of the Loa22 and mutation variants. The molecular mass of Loa22 protein was calculated as 22 kDa and the SDS-PAGE showed the single band slightly higher than 25 kDa. (E) Western blot of purified Loa22WT and variants. The Loa22 WT and mutation variants were recognized by anti 6XHis tag antibody. (F) Size exclusion chromatography of purified Loa22 protein. The single peak of purified Loa22 protein with molecular mass about 22 kDa indicated that the protein is a uniform conformation. (G) LAL test of the purified recombinant Loa22. The endotoxin of the recombinant protein was assay by LAL test to measure the endotoxin contamination.* p<0.05; *, p<0.01;, p<0.001.

Article Snippet: The human TLR2 gene was sub-cloned from plasmid pUNO-TLR2 (Invivogen, San Diego, CA) and inserted into a lentivirus expression vector with a V5 tag at the C-terminus.

Techniques: Purification, Sequencing, Binding Assay, Mutagenesis, SDS Page, Western Blot, Size-exclusion Chromatography, Recombinant

Journal: bioRxiv

Article Title: Peptidoglycan Mediates Loa22 and Toll-like Receptor 2 Interactions in Pathogenic Leptospira

doi: 10.1101/520288

Figure Lengend Snippet: Loa22 co-localized with TLR2 on HEK293-TLR2 cells. The purified rLoa22 protein and its mutation variants with LPGN were incubated with HEK293-TLR2 cell for 4 h and the cell were fixed for confocal microscopy measurement. (A) rLoa22WT-LPGNwas incubated with HEK293 cell. (B) rLoa22WT-LPGNwas incubated with HEK293-TLR2 cell. (C) rLoa22D122A-LPGN was incubated with HEK293-TLR2 cell (D) rLoa22R143A-LPGN was incubated with HEK293-TLR2 cell. The nucleus was stained by DAPI (blue), TLR2 was stained by Alexa488 (green), and Loa22 was stained by Alexa594 (red). The yellow color indicated that the two proteins were co-localized in HEK293-TLR2 cell.

Article Snippet: The human TLR2 gene was sub-cloned from plasmid pUNO-TLR2 (Invivogen, San Diego, CA) and inserted into a lentivirus expression vector with a V5 tag at the C-terminus.

Techniques: Purification, Mutagenesis, Incubation, Confocal Microscopy, Staining

Journal: bioRxiv

Article Title: Peptidoglycan Mediates Loa22 and Toll-like Receptor 2 Interactions in Pathogenic Leptospira

doi: 10.1101/520288

Figure Lengend Snippet: Inflammatory responses induced by rLoa22 protein in HEK293-TLR2 cell. The downstream inflammatory responses from TLR2 signaling such CXCL8/IL8, hCCL2/MCP-1, and hTNF-α were measured 2 hours after adding the stimulation agents. (A) Recombinant Loa22 isolated from Clearcoli TM E. coli contained low level of endotoxin contamination and used for cytokines stimulation assay. LipL32 protein was used as positive control. The His6-Tag of rLoa22 was removed by Enterokinase protease. rLoa22 was treated with heat (100 ° C, 30 min) and proteinase K (20 μg/ml at 63 °C for 18 h) to denature and digested the protein. (B) Inflammatory responses stimulated by Loa22 and its variants in the presence or absence of PGN. rLoa22WT and mutated variants, rLoa22D122A and rLoa22R143A, were used to treat with HEK293-TLR2 cell in the presence or absence of LPGN.* p<0.05; *, p<0.01; *, p<0.001.

Article Snippet: The human TLR2 gene was sub-cloned from plasmid pUNO-TLR2 (Invivogen, San Diego, CA) and inserted into a lentivirus expression vector with a V5 tag at the C-terminus.

Techniques: Recombinant, Isolation, Positive Control

Journal: bioRxiv

Article Title: Peptidoglycan Mediates Loa22 and Toll-like Receptor 2 Interactions in Pathogenic Leptospira

doi: 10.1101/520288

Figure Lengend Snippet: In vitro assay of the interaction between TLR2 and rLoa22-LPGN complexes. (A) Purification of TLR2 from HEK293-TLR2 cell. Left panel, SDS page of purified TLR2; right panel, western blot of purified TLR2. (B) Functional assays of purified TLR2 to Pam 3 CSK 4 and Leptospira LipL32. (C) ELISA assay of the interaction between TLR2 and rLoa22. (D) AFM force-distance curves of the interaction between TLR2 and rLoa22. (E) The binding frequency of the interaction between TLR2 and rLoa22. TLR2 interacted to rLoa22WT and mutation variants, rLoa22D122A and rLoa22R143A in the presence or absence of LPGN. BSA was used as negative control and LipL32 was used as positive control.* p<0.05;, p<0.01; *, p<0.001.

Article Snippet: The human TLR2 gene was sub-cloned from plasmid pUNO-TLR2 (Invivogen, San Diego, CA) and inserted into a lentivirus expression vector with a V5 tag at the C-terminus.

Techniques: In Vitro, Purification, SDS Page, Western Blot, Functional Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Mutagenesis, Negative Control, Positive Control

Journal: bioRxiv

Article Title: Peptidoglycan Mediates Loa22 and Toll-like Receptor 2 Interactions in Pathogenic Leptospira

doi: 10.1101/520288

Figure Lengend Snippet: TLR2 downstream signaling cascades assays after rLoa22-LPGN complexes stimulation. (A) rLoa22-LPGN complexes stimulated HEK293 or HEK293-TLR2 cells for 24h and downstream signaling cascades were assayed. (B) rLoa22-LPGN complexes stimulated HK2 cells in the presence and absence of TLR2 antibody for 24h and downstream signaling cascades were assayed.

Article Snippet: The human TLR2 gene was sub-cloned from plasmid pUNO-TLR2 (Invivogen, San Diego, CA) and inserted into a lentivirus expression vector with a V5 tag at the C-terminus.

Techniques:

Journal: bioRxiv

Article Title: Peptidoglycan Mediates Loa22 and Toll-like Receptor 2 Interactions in Pathogenic Leptospira

doi: 10.1101/520288

Figure Lengend Snippet: Characterization and bioinformatic analysis of Loa22 protein. (A) Domains prediction of Loa22. Loa22 contains 195 amino acids and the domains preditions revealed that the N terminal contains signal peptide ( – ) and C terminal of contains OmpA_C like domain (78-186). (B) Immuno-precipitant of purified TLR2 protein and Loa22 protein. Anti-TLR2, anti-LipL32 and anti-Loa22 antibodies were used to detect the presence of these proteins. (C) Sequence alignment of Loa22. Loa22 sequence was aligned with other OmpA domain protein including ABOmpA and Pal protein from A. baumannii and E. coli, respectively. Two pivotal residues (Asp122 and Arg143; red arrows) were highly conserved in Loa22 and other OmpA domain proteins and the two residues were responsible for peptidylglycan (PGN) binding. (D) Purification of the Loa22 and mutation variants. The molecular mass of Loa22 protein was calculated as 22 kDa and the SDS-PAGE showed the single band slightly higher than 25 kDa. (E) Western blot of purified Loa22WT and variants. The Loa22 WT and mutation variants were recognized by anti 6XHis tag antibody. (F) Size exclusion chromatography of purified Loa22 protein. The single peak of purified Loa22 protein with molecular mass about 22 kDa indicated that the protein is a uniform conformation. (G) LAL test of the purified recombinant Loa22. The endotoxin of the recombinant protein was assay by LAL test to measure the endotoxin contamination.* p<0.05; *, p<0.01;, p<0.001.

Article Snippet: The vector containing human tlr2 gene (pUNO-hTLR2) was obtained from InvivoGen for gene manipulation.

Techniques: Purification, Sequencing, Binding Assay, Mutagenesis, SDS Page, Western Blot, Size-exclusion Chromatography, Recombinant

Journal: bioRxiv

Article Title: Peptidoglycan Mediates Loa22 and Toll-like Receptor 2 Interactions in Pathogenic Leptospira

doi: 10.1101/520288

Figure Lengend Snippet: Loa22 co-localized with TLR2 on HEK293-TLR2 cells. The purified rLoa22 protein and its mutation variants with LPGN were incubated with HEK293-TLR2 cell for 4 h and the cell were fixed for confocal microscopy measurement. (A) rLoa22WT-LPGNwas incubated with HEK293 cell. (B) rLoa22WT-LPGNwas incubated with HEK293-TLR2 cell. (C) rLoa22D122A-LPGN was incubated with HEK293-TLR2 cell (D) rLoa22R143A-LPGN was incubated with HEK293-TLR2 cell. The nucleus was stained by DAPI (blue), TLR2 was stained by Alexa488 (green), and Loa22 was stained by Alexa594 (red). The yellow color indicated that the two proteins were co-localized in HEK293-TLR2 cell.

Article Snippet: The vector containing human tlr2 gene (pUNO-hTLR2) was obtained from InvivoGen for gene manipulation.

Techniques: Purification, Mutagenesis, Incubation, Confocal Microscopy, Staining

Journal: bioRxiv

Article Title: Peptidoglycan Mediates Loa22 and Toll-like Receptor 2 Interactions in Pathogenic Leptospira

doi: 10.1101/520288

Figure Lengend Snippet: Inflammatory responses induced by rLoa22 protein in HEK293-TLR2 cell. The downstream inflammatory responses from TLR2 signaling such CXCL8/IL8, hCCL2/MCP-1, and hTNF-α were measured 2 hours after adding the stimulation agents. (A) Recombinant Loa22 isolated from Clearcoli TM E. coli contained low level of endotoxin contamination and used for cytokines stimulation assay. LipL32 protein was used as positive control. The His6-Tag of rLoa22 was removed by Enterokinase protease. rLoa22 was treated with heat (100 ° C, 30 min) and proteinase K (20 μg/ml at 63 °C for 18 h) to denature and digested the protein. (B) Inflammatory responses stimulated by Loa22 and its variants in the presence or absence of PGN. rLoa22WT and mutated variants, rLoa22D122A and rLoa22R143A, were used to treat with HEK293-TLR2 cell in the presence or absence of LPGN.* p<0.05; *, p<0.01; *, p<0.001.

Article Snippet: The vector containing human tlr2 gene (pUNO-hTLR2) was obtained from InvivoGen for gene manipulation.

Techniques: Recombinant, Isolation, Positive Control

Journal: bioRxiv

Article Title: Peptidoglycan Mediates Loa22 and Toll-like Receptor 2 Interactions in Pathogenic Leptospira

doi: 10.1101/520288

Figure Lengend Snippet: In vitro assay of the interaction between TLR2 and rLoa22-LPGN complexes. (A) Purification of TLR2 from HEK293-TLR2 cell. Left panel, SDS page of purified TLR2; right panel, western blot of purified TLR2. (B) Functional assays of purified TLR2 to Pam 3 CSK 4 and Leptospira LipL32. (C) ELISA assay of the interaction between TLR2 and rLoa22. (D) AFM force-distance curves of the interaction between TLR2 and rLoa22. (E) The binding frequency of the interaction between TLR2 and rLoa22. TLR2 interacted to rLoa22WT and mutation variants, rLoa22D122A and rLoa22R143A in the presence or absence of LPGN. BSA was used as negative control and LipL32 was used as positive control.* p<0.05;, p<0.01; *, p<0.001.

Article Snippet: The vector containing human tlr2 gene (pUNO-hTLR2) was obtained from InvivoGen for gene manipulation.

Techniques: In Vitro, Purification, SDS Page, Western Blot, Functional Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Mutagenesis, Negative Control, Positive Control

Journal: bioRxiv

Article Title: Peptidoglycan Mediates Loa22 and Toll-like Receptor 2 Interactions in Pathogenic Leptospira

doi: 10.1101/520288

Figure Lengend Snippet: TLR2 downstream signaling cascades assays after rLoa22-LPGN complexes stimulation. (A) rLoa22-LPGN complexes stimulated HEK293 or HEK293-TLR2 cells for 24h and downstream signaling cascades were assayed. (B) rLoa22-LPGN complexes stimulated HK2 cells in the presence and absence of TLR2 antibody for 24h and downstream signaling cascades were assayed.

Article Snippet: The vector containing human tlr2 gene (pUNO-hTLR2) was obtained from InvivoGen for gene manipulation.

Techniques:

Journal: PLoS Pathogens

Article Title: Lyn Delivers Bacteria to Lysosomes for Eradication through TLR2-Initiated Autophagy Related Phagocytosis

doi: 10.1371/journal.ppat.1005363

Figure Lengend Snippet: (A) MH-S cells were infected by PAO1 (MOI = 10), HKPa (MOI = 10) and Pa LPS (100 ng/ml) for 1 h. HKPa was obtained by heating PAO1 at 60°C for 60 min. Cell lysates were collected and immunoblotting of pLyn, Lyn and LC3 were performed. (B, C) MH-S cells were transfected with LC3-RFP plasmid for 24 h. CLSM imaging showed infection by PAO1, HKPa and Pa LPS induced LC3 puncta. LC3 puncta in each cell were counted. Data are derived from 100 cells in each sample. (D) MH-S cells were transfected with Ctrl, TLR2 or TLR4 siRNA for 24 h, respectively. Immunoblotting were performed to prove the knock down of TLR2 or TLR4. (E, F) After TLR2 or TLR4 knock down as above, the cells were infected with PAO1 (MOI = 10, 1 h). MTT assays were used to measure the cell viability and CFU assays were used to measure phagocytosis. (G, H) MH-S cells were co-transfected with LC3-RFP and Ctrl, TLR2 or TLR4 siRNA for 24 h, respectively. Cells were then infected with PAO1 as above. CLSM imaging showed infection-induced LC3 puncta. LC3 puncta in each cell were counted. Data are derived from 100 cells in each sample. (I) MH-S cells were transfected with Ctrl or TLR2 siRNA for 24 h, respectively. Cells were infected with PAO1 as above. Cell lysates were performed for immunoblotting to detect pLyn, Lyn and LC3. (J) The HEK293 cells were stably transfected with a pUNO-TLR2 plasmid which expresses TLR2. TLR2 stable expression cells and 293/null cells were infected with PAO1 as above. Immunoblotting was performed to detect TLR2, pLyn, Lyn and LC3. (K, L) 293 cells were transiently transfected with LC3-RFP. After 24 hours cells were infected with PAO1-GFP (MOI = 10, 1 h). Arrows indicate significant LC3 puncta upon Pa infection using CLSM imaging. LC3 puncta in each cell were counted and the percentage of LC3 + /Pa + events (cell with colocalized puncta of LC3-RFP and PAO1-GFP) is shown. Data are derived from 100 cells in each sample. (M, N) MH-S cells were pretreated with Pam 3 CSK 4 (300 ng/ml), and infected with PAO1 as above. Cells were homogenized for CFU and immunoblotting to detect TLR2, pLyn, Lyn and LC3. All data are representative as means+SD of three independent experiments. One-way ANOVA (Tukey’s post hoc); *, p<0.05; **, p<0.01. Scale bar = 5 μm.

Article Snippet: Stable TLR2 expression HEK293 cells (ATCC CRL-157) using a pUNO-TLR2 plasmid were obtained from InvivoGen (San Diego, CA) and cultured in DMEM medium as above.

Techniques: Infection, Western Blot, Transfection, Plasmid Preparation, Imaging, Derivative Assay, Stable Transfection, Expressing