Journal: The Journal of Biological Chemistry

Article Title: Functional binding of E-selectin to its ligands is enhanced by structural features beyond its lectin domain

doi: 10.1074/jbc.RA119.010910

Figure Lengend Snippet: Expression and purification of functional E-selectin proteins. A , schematic representation of E-selectin constructs. i , E-S6-IgG consisting of the structural domains of a commonly used E-selectin-IgG/Fc (dimer) chimera recombinant protein. ii , monomeric version of full-length E-selectin (E-S6). iii and iv , truncated E-selectin formed by domain deletion of either the last four SCRs, producing constructs with only the first two SCRs (E-S2), or deletion of all of the SCRs, yielding a minimal construct possessing only the main domains for binding (E-S0). Several tags were included at the C terminus of each construct, such as a histidine tag (double His 6 or a single His 8 ) and a Strep-tag, to facilitate the purification and subsequent immobilization of E-selectins (note that an Avi tag located after the EGF domain in E-S6-IgG, E-S2, and E-S0 and a TEV tag included before His 8 in E-S6 are omitted from this diagram as they were not addressed in this study). B , analytical native PAGE of the recombinant E-selectin proteins. E-S6-IgG, E-S6, E-S2, E-S2-A28H, and E-S0 were diluted in 1× Native PAGE buffer, loaded on a 10% TBE gel and run in 1× Tris-glycine buffer. The majority of molecules in each recombinant protein sample appear as a single species, with a minor amount of oligomerization in case of E-S6; the majority of molecules in E-S6-IgG appear as a dimer through the C terminus Fc region, with some slightly monomeric versions appearing as a faint band of lower molecular weight.

Article Snippet: The KG1a cells (at a concentration of 1 × 10 6 cells/ml) were stained with 10 μg/ml of each of the E-selectins, E-S6-IgG, E-S6, E-S2, E-S2-A28H, or E-S0, in 100 μl HBSS with 5% FBS, in the presence of either 2 m m CaCl 2 or 20 m m EDTA, at 4 °C for 20 min. Next, the stained cell samples were washed with the same HBSS buffer (containing either 2 m m CaCl 2 or 20 m m EDTA) one time, and further stained with 10 μg/ml of either Strep-tag II mAb (EMD Millipore), mouse IgG1 PE conjugated isotype control antibody (R&D Systems), or PE rat anti-mouse IgG1 (Clone A85–1) (BD Biosciences) as the secondary control, and then were incubated at 4 °C for 20 min.

Techniques: Expressing, Purification, Functional Assay, Construct, Recombinant, Binding Assay, Strep-tag, Clear Native PAGE, Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: Functional binding of E-selectin to its ligands is enhanced by structural features beyond its lectin domain

doi: 10.1074/jbc.RA119.010910

Figure Lengend Snippet: Flow cytometric and Western blotting analyses comparing the binding functionalities of the various forms of E-selectin proteins to KG1a cells. A , E-S6-IgG, E-S6, E-S2, E-S2-A28H, and E-S0 were tested for their ability to bind to ligands on KG1a cells. Mouse monoclonal anti-Strep antibody (followed by a fluorescently labeled antibody against anti-Strep) was used to detect the E-selectin protein bound to ligands on the surface of KG1a cells in the presence of calcium ( green histogram) or EDTA ( red histogram). Samples stained with only the secondary antibody are included as a control to determine secondary antibody specificity toward mouse monoclonal anti-Strep antibody ( blue histogram). B and C , the percentages of KG1a cells bound to each of the E-selectin proteins and the geometric means of the fluorescence signals were determined from n = 3 independent experiments ( n = 3 ; a , indicates significance compared with E-S6-IgG; b , indicates significance compared with E-S6; c , indicates significance compared with E-S2; and d , indicates significance compared with E-S2-A28H, p ≤ 0.05) and the means are depicted as mean ± S.E.M. in ( B ) and ( C ), respectively. D , Western blot analysis of E-selectin protein binding to immunoprecipitated PSGL-1 and CD44. KG1a lysates were prepared, and PSGL-1 and CD44 were immunoprecipitated and subjected to the Western blot analysis. The resulting blots ( upper panel , CD44; lower panel , PSGL-1) were stained with 1 μg/ml of E-S6-IgG, E-S6, E-S2, or E-S0, as indicated in the figure, in the presence of calcium. Anti-strep mAb was used against bound E-selectin for subsequent chemiluminescence detection using HRP-conjugated anti-mouse IgG. Blots stained in the presence of EDTA to confirm binding specificity showed no binding activity ( Fig. S4 ).

Article Snippet: The KG1a cells (at a concentration of 1 × 10 6 cells/ml) were stained with 10 μg/ml of each of the E-selectins, E-S6-IgG, E-S6, E-S2, E-S2-A28H, or E-S0, in 100 μl HBSS with 5% FBS, in the presence of either 2 m m CaCl 2 or 20 m m EDTA, at 4 °C for 20 min. Next, the stained cell samples were washed with the same HBSS buffer (containing either 2 m m CaCl 2 or 20 m m EDTA) one time, and further stained with 10 μg/ml of either Strep-tag II mAb (EMD Millipore), mouse IgG1 PE conjugated isotype control antibody (R&D Systems), or PE rat anti-mouse IgG1 (Clone A85–1) (BD Biosciences) as the secondary control, and then were incubated at 4 °C for 20 min.

Techniques: Western Blot, Binding Assay, Labeling, Staining, Fluorescence, Protein Binding, Immunoprecipitation, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Functional binding of E-selectin to its ligands is enhanced by structural features beyond its lectin domain

doi: 10.1074/jbc.RA119.010910

Figure Lengend Snippet: Glycan microarray analysis. A , upper panel , E-selectins (E-S6-IgG, E-S6, E-S2 (WT), and E-S0) were introduced to 4× glycan subarray slides, and each bound construct was detected with biotin anti-His followed by Cy3 Equivalent Dye-Streptavidin. The layout of each array is shown with the location of each glycan, positive (+) and negative (−) controls. B , fluorescent intensity of each glycan was determined from quadruplicate samples, and a bar chart was generated and the means are depicted as mean ± S.E.M. for each construct. Each glycan is represented as a number and a color on the x -axis. C , a subset of the fluorescent intensity highlighting the differences in binding of each E-selectin to Neu5Ac-α-2,3-Gal-β-1,4-(Fuc-α-1,3)-GlcNAc-β- (sialyl-Lewis X)-Sp (33: magenta ), Neu5Ac-α-2,3-Gal-β-1,3-(Fuc-α-1,4)-GlcNAc-β-(sialyl-Lewis A)-Sp (34: green ), and Neu5Gc-α-2,3-Gal-β-1,3-(Fuc-α-1,4)-GlcNAc-β-(sialyl-Lewis A)-Sp (35: peach ). OCH 2 CH 2 Ch 2 NH 2 and NH(CH 3 )OCH 2 CH 2 NH 2 linkers are represented as Sp and Sp1, respectively.

Article Snippet: The KG1a cells (at a concentration of 1 × 10 6 cells/ml) were stained with 10 μg/ml of each of the E-selectins, E-S6-IgG, E-S6, E-S2, E-S2-A28H, or E-S0, in 100 μl HBSS with 5% FBS, in the presence of either 2 m m CaCl 2 or 20 m m EDTA, at 4 °C for 20 min. Next, the stained cell samples were washed with the same HBSS buffer (containing either 2 m m CaCl 2 or 20 m m EDTA) one time, and further stained with 10 μg/ml of either Strep-tag II mAb (EMD Millipore), mouse IgG1 PE conjugated isotype control antibody (R&D Systems), or PE rat anti-mouse IgG1 (Clone A85–1) (BD Biosciences) as the secondary control, and then were incubated at 4 °C for 20 min.

Techniques: Microarray, Construct, Generated, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Functional binding of E-selectin to its ligands is enhanced by structural features beyond its lectin domain

doi: 10.1074/jbc.RA119.010910

Figure Lengend Snippet: Cell rolling analysis of KG1a cells on immobilized E-selectin proteins. A , schematic representation of the experimental steps involved in the microfluidics-based flow assay. To ensure the uniform immobilization of each of the proteins, identical concentrations of protein A were deposited on each channel of an uncoated μ slide VI 0.1 microscopy chamber. This was followed by the addition of anti-histidine antibody and, subsequently, E-selectin proteins were introduced and captured through their C-terminal histidine tags (refer to Fig. S6 : each E-selectin can be pulled down equivalently using anti-histidine). KG1a cells in perfusion buffer containing 0.5 m m Ca 2+ were drawn into the E-selectin–coated channels at various shear stresses (1 dyne/cm 2 , 2 dyne/cm 2 , 3 dyne/cm 2 , 4 dyne/cm 2 , 5 dyne/cm 2 , and 6 dyne/cm 2 ) for duration of 30 s each. B , adhesion bar graph of rolling velocities (μm/s) from n = 3 independent experiments, evaluating the rolling of KG1a on histidine-immobilized E-selectins in the presence of 0.5 m m Ca 2+ , represented as mean ± S.E.M. C , bar graph representing the number of KG1a cells rolling on each construct as an alternative measurement for adhesive strength from n = 3 independent experiments, represented as mean ± S.E.M. ( n = 3 ; a indicates significance compared with E-S6-IgG, b indicates significance compared with E-S6, c indicates significance compared with E-S2, and d indicates significance compared with E-S2-A28H, p ≤ 0.05).

Article Snippet: The KG1a cells (at a concentration of 1 × 10 6 cells/ml) were stained with 10 μg/ml of each of the E-selectins, E-S6-IgG, E-S6, E-S2, E-S2-A28H, or E-S0, in 100 μl HBSS with 5% FBS, in the presence of either 2 m m CaCl 2 or 20 m m EDTA, at 4 °C for 20 min. Next, the stained cell samples were washed with the same HBSS buffer (containing either 2 m m CaCl 2 or 20 m m EDTA) one time, and further stained with 10 μg/ml of either Strep-tag II mAb (EMD Millipore), mouse IgG1 PE conjugated isotype control antibody (R&D Systems), or PE rat anti-mouse IgG1 (Clone A85–1) (BD Biosciences) as the secondary control, and then were incubated at 4 °C for 20 min.

Techniques: Microscopy, Construct

Journal: The Journal of Biological Chemistry

Article Title: Functional binding of E-selectin to its ligands is enhanced by structural features beyond its lectin domain

doi: 10.1074/jbc.RA119.010910

Figure Lengend Snippet: Rate constants for E-selectin protein binding to CD44/HCELL. A , experimental schematic diagram of the SPR-based real-time binding assay. Step 1, mAb immobilization on CM5 chip; step 2, KG1a lysate injection to capture CD44/HCELL; step 3, injection of various E-selectin proteins. B , binding of titrated concentrations of E-S6-IgG to captured CD44/HCELL from KG1a lysates via immobilized Hermes-3 (4113 RU); sensorgram representation of sequential injections of E-S6-IgG at 0.005, 0.01, 0.02, 0.05, 0.09, 0.19, 0.38, 0.75, 1.5, and 3 μ m at a flow rate of 20 μl/min for 90 s each, spaced by a 60-s washing step. The lysate injection is not shown. The sensorgram profile was corrected for nonspecific interaction by subtracting isotype control (4466 RU). K D and k off values were determined, as described previously . k on was calculated using K D and k off values. C , binding of E-S6 (monomer form of the full-length E-selectin) to CD44/HCELL injected at different concentrations, 0.1, 0.19, 0.38, 0.77, 1.53, 3.06, 6.12, 12.25, 24.49, and 48.98 μ m , using similar experimental conditions to those described in ( B ). The RU for Hermes-3 and its isotype control were 8670 and 6770, respectively. D , binding of truncated E-S2 to CD44/HCELL injected consecutively at concentrations 0.04, 0.08, 0.15, 0.31, 0.62, 1.24, 2.48, 4.95, 9.9, and 19.8 μ m using similar experimental conditions to ( B ). The RU for Hermes-3 and its isotype control were 8450 and 5090, respectively. E , binding of titrated concentrations of E-S2-A28H to captured CD44/HCELL at different concentrations, 0.2, 0.3, 0.7, 1.3, 2.6, 5.2, 10.5, and 20.9 μ m , using similar experimental conditions to ( B ). The RU for Hermes-3 and its isotype control were 6680 and 6360, respectively. F , binding of the SCR-deficient E-S0 to CD44/HCELL injected consecutively at concentrations of 0.14, 0.27, 0.56, 1.09, 2.19, 4.38, 8.75, 17.50, 35, and 70 μ m using similar experimental conditions to ( B ). The RU for Hermes-3 and its isotype control were 6200 and 4900, respectively. G , table summarizing the binding constants of the E-selectin proteins to CD44/HCELL from n = 3 independent experiments for each E-selectin ( B–F ), reported as mean ± S.E.M. ( n = 3 , a indicates significance compared with E-S6-IgG, b indicates significance compared with E-S6, c indicates significance compared with E-S2, and d indicates significance compared with E-S2-A28H, p ≤ 0.05).

Article Snippet: The KG1a cells (at a concentration of 1 × 10 6 cells/ml) were stained with 10 μg/ml of each of the E-selectins, E-S6-IgG, E-S6, E-S2, E-S2-A28H, or E-S0, in 100 μl HBSS with 5% FBS, in the presence of either 2 m m CaCl 2 or 20 m m EDTA, at 4 °C for 20 min. Next, the stained cell samples were washed with the same HBSS buffer (containing either 2 m m CaCl 2 or 20 m m EDTA) one time, and further stained with 10 μg/ml of either Strep-tag II mAb (EMD Millipore), mouse IgG1 PE conjugated isotype control antibody (R&D Systems), or PE rat anti-mouse IgG1 (Clone A85–1) (BD Biosciences) as the secondary control, and then were incubated at 4 °C for 20 min.

Techniques: Protein Binding, Binding Assay, Injection

Journal: iScience

Article Title: TLR2/NF-κB signaling in macrophage/microglia mediated COVID-pain induced by SARS-CoV-2 envelope protein

doi: 10.1016/j.isci.2024.111027

Figure Lengend Snippet: Identification of several coat proteins of SARS-COV-2 in human tissues (A, C, E, and G) Immunostaining detected the presence of S2E, S2S, and S2M (red) in the lung, SDH, DRG, and TG from COVID-19 infected donors, but not in the uninfected tissues (blue for DAPI). Scale bar: 50 μm. (B, D, F, and H) Quantitative analysis of S2E, S2S, and S2M positive area percentages in lung, SDH, DRG and TG from infected donors. ∗ p < 0.05 , ∗∗ p < 0.01 , ∗∗∗ p < 0.001 , Student’s t test.

Article Snippet: The used primary antibodies included: rabbit anti-TLR2, 1:200, Abcam (ab209216); mouse Anti-His-tag, 1:200, MBL (D291-3); goat anti-IBA1, 1:400, Abcam (ab5076); Chicken anti-GFAP, 1:400, Abcam (ab4674); mouse anti-NeuN, 1:1000, Abcam (ab104224); mouse anti-c-Fos, 1:1000, Abcam (ab208942); rabbit anti-p-ERK, 1:400, CST (4370); rabbit anti-S2E, 1:500, Proteintech (28904); rabbit anti-S2S, 1:500, GeneTex (GTX635654); rabbit anti-S2M, 1:500, GeneTex (GTX636245); rabbit mAb isotype IgG, 1:500, CST(3900).

Techniques: Immunostaining, Infection

Journal: iScience

Article Title: TLR2/NF-κB signaling in macrophage/microglia mediated COVID-pain induced by SARS-CoV-2 envelope protein

doi: 10.1016/j.isci.2024.111027

Figure Lengend Snippet: The algesiogenic effects of SARS-CoV-2 viral proteins via i.d. injection (A–D) Paw withdrawal frequency to 0.4 g von Frey filament and withdrawal latency in Hargreaves test of male and female mice after i.d. injection of S2E with gradually increasing doses (0.01 μg, 0.1 μg, and 1 μg). n = 6 mice/group. # p < 0.05 , && p < 0.01 , &&& or ### p < 0.001 , ∗∗∗∗ or #### or &&&& p < 0.0001 vs. PBS, two-way ANOVA for repeated measures followed by Dunnett’s multiple comparisons test. (BL: Baseline). (E–H) Paw withdrawal frequency to 0.4 g von Frey filament and latency in male and female mice after i.d. application of PBS, S2E, S2S, and S2M (1 μg). n = 6 mice/group; ∗ p < 0.05 , ∗∗ p < 0.01 , ∗∗∗ p < 0.001 vs. PBS, two-way ANOVA for repeated measures followed by Dunnett’s post hoc test. (BL: Baseline). (I and J) Immunofluorescence of p -ERK (green) in DRG from mice receiving i.d. injection of PBS or S2E, along with quantitative analysis (blue for DAPI). Scale bar: 50 μm n = 3 mice/group. ∗∗ p < 0.01 , ∗∗∗∗ p < 0.0001 , Student’s t test. (K and L) Immunofluorescence of c-Fos (red) in SDH from mice receiving i.d. injection of PBS or S2E (blue for DAPI), along with quantitative analysis. Scale bar: 50 μm n = 3 mice/group. ∗∗∗ p < 0.001 , ∗∗∗∗ p < 0.0001 , Student’s t test.

Article Snippet: The used primary antibodies included: rabbit anti-TLR2, 1:200, Abcam (ab209216); mouse Anti-His-tag, 1:200, MBL (D291-3); goat anti-IBA1, 1:400, Abcam (ab5076); Chicken anti-GFAP, 1:400, Abcam (ab4674); mouse anti-NeuN, 1:1000, Abcam (ab104224); mouse anti-c-Fos, 1:1000, Abcam (ab208942); rabbit anti-p-ERK, 1:400, CST (4370); rabbit anti-S2E, 1:500, Proteintech (28904); rabbit anti-S2S, 1:500, GeneTex (GTX635654); rabbit anti-S2M, 1:500, GeneTex (GTX636245); rabbit mAb isotype IgG, 1:500, CST(3900).

Techniques: Injection, Immunofluorescence

Journal: iScience

Article Title: TLR2/NF-κB signaling in macrophage/microglia mediated COVID-pain induced by SARS-CoV-2 envelope protein

doi: 10.1016/j.isci.2024.111027

Figure Lengend Snippet: The algesiogenic effects of SARS-CoV-2 viral proteins via i.t. injection (A–D) Paw withdrawal frequency to 0.4 g von Frey filament and withdrawal latency in Hargreaves test of male and female mice after i.t. injection of S2E with gradually increasing doses (0.01 μg, 0.1 μg and 1 μg). n = 6 mice/group. # or & p < 0.05 , && p < 0.01 , ∗∗∗ or ### or &&& p < 0.001 , ∗∗∗∗ or #### or &&&& p < 0.0001 vs. PBS, two-way ANOVA for repeated measures followed by Dunnett’s multiple comparisons test. (BL: Baseline). (E–H) Paw withdrawal frequency and latency in male and female mice after i.t. application of PBS, S2E, S2S, and S2M (1 μg). n = 6 mice/group; ∗ p < 0.05 , ∗∗ p < 0.01 , ∗∗∗∗ p < 0.0001 vs. PBS, two-way ANOVA for repeated measures followed by Dunnett’s post hoc test. (BL: Baseline). (I and J) Immunofluorescence of c-Fos (red) in SDH from mice receiving i.t. injection of PBS or S2E (blue for DAPI), along with quantitative analysis. Scale bar: 50 μm n = 3 mice/group. ∗∗∗∗ p < 0.0001 , Student’s t test.

Article Snippet: The used primary antibodies included: rabbit anti-TLR2, 1:200, Abcam (ab209216); mouse Anti-His-tag, 1:200, MBL (D291-3); goat anti-IBA1, 1:400, Abcam (ab5076); Chicken anti-GFAP, 1:400, Abcam (ab4674); mouse anti-NeuN, 1:1000, Abcam (ab104224); mouse anti-c-Fos, 1:1000, Abcam (ab208942); rabbit anti-p-ERK, 1:400, CST (4370); rabbit anti-S2E, 1:500, Proteintech (28904); rabbit anti-S2S, 1:500, GeneTex (GTX635654); rabbit anti-S2M, 1:500, GeneTex (GTX636245); rabbit mAb isotype IgG, 1:500, CST(3900).

Techniques: Injection, Immunofluorescence

Journal: iScience

Article Title: TLR2/NF-κB signaling in macrophage/microglia mediated COVID-pain induced by SARS-CoV-2 envelope protein

doi: 10.1016/j.isci.2024.111027

Figure Lengend Snippet:

Article Snippet: The used primary antibodies included: rabbit anti-TLR2, 1:200, Abcam (ab209216); mouse Anti-His-tag, 1:200, MBL (D291-3); goat anti-IBA1, 1:400, Abcam (ab5076); Chicken anti-GFAP, 1:400, Abcam (ab4674); mouse anti-NeuN, 1:1000, Abcam (ab104224); mouse anti-c-Fos, 1:1000, Abcam (ab208942); rabbit anti-p-ERK, 1:400, CST (4370); rabbit anti-S2E, 1:500, Proteintech (28904); rabbit anti-S2S, 1:500, GeneTex (GTX635654); rabbit anti-S2M, 1:500, GeneTex (GTX636245); rabbit mAb isotype IgG, 1:500, CST(3900).

Techniques: Recombinant, Protein Extraction, Endotoxin Assay, Western Blot, Virus, Software, Real-time Polymerase Chain Reaction, Imaging

Journal: iScience

Article Title: TLR2/NF-κB signaling in macrophage/microglia mediated COVID-pain induced by SARS-CoV-2 envelope protein

doi: 10.1016/j.isci.2024.111027

Figure Lengend Snippet: Identification of several coat proteins of SARS-COV-2 in human tissues (A, C, E, and G) Immunostaining detected the presence of S2E, S2S, and S2M (red) in the lung, SDH, DRG, and TG from COVID-19 infected donors, but not in the uninfected tissues (blue for DAPI). Scale bar: 50 μm. (B, D, F, and H) Quantitative analysis of S2E, S2S, and S2M positive area percentages in lung, SDH, DRG and TG from infected donors. ∗ p < 0.05 , ∗∗ p < 0.01 , ∗∗∗ p < 0.001 , Student’s t test.

Article Snippet: The used primary antibodies included: rabbit anti-TLR2, 1:200, Abcam (ab209216); mouse Anti-His-tag, 1:200, MBL (D291-3); goat anti-IBA1, 1:400, Abcam (ab5076); Chicken anti-GFAP, 1:400, Abcam (ab4674); mouse anti-NeuN, 1:1000, Abcam (ab104224); mouse anti-c-Fos, 1:1000, Abcam (ab208942); rabbit anti-p-ERK, 1:400, CST (4370); rabbit anti-S2E, 1:500, Proteintech (28904); rabbit anti-S2S, 1:500, GeneTex (GTX635654); rabbit anti-S2M, 1:500, GeneTex (GTX636245); rabbit mAb isotype IgG, 1:500, CST(3900).

Techniques: Immunostaining, Infection

Journal: iScience

Article Title: TLR2/NF-κB signaling in macrophage/microglia mediated COVID-pain induced by SARS-CoV-2 envelope protein

doi: 10.1016/j.isci.2024.111027

Figure Lengend Snippet: The algesiogenic effects of SARS-CoV-2 viral proteins via i.d. injection (A–D) Paw withdrawal frequency to 0.4 g von Frey filament and withdrawal latency in Hargreaves test of male and female mice after i.d. injection of S2E with gradually increasing doses (0.01 μg, 0.1 μg, and 1 μg). n = 6 mice/group. # p < 0.05 , && p < 0.01 , &&& or ### p < 0.001 , ∗∗∗∗ or #### or &&&& p < 0.0001 vs. PBS, two-way ANOVA for repeated measures followed by Dunnett’s multiple comparisons test. (BL: Baseline). (E–H) Paw withdrawal frequency to 0.4 g von Frey filament and latency in male and female mice after i.d. application of PBS, S2E, S2S, and S2M (1 μg). n = 6 mice/group; ∗ p < 0.05 , ∗∗ p < 0.01 , ∗∗∗ p < 0.001 vs. PBS, two-way ANOVA for repeated measures followed by Dunnett’s post hoc test. (BL: Baseline). (I and J) Immunofluorescence of p -ERK (green) in DRG from mice receiving i.d. injection of PBS or S2E, along with quantitative analysis (blue for DAPI). Scale bar: 50 μm n = 3 mice/group. ∗∗ p < 0.01 , ∗∗∗∗ p < 0.0001 , Student’s t test. (K and L) Immunofluorescence of c-Fos (red) in SDH from mice receiving i.d. injection of PBS or S2E (blue for DAPI), along with quantitative analysis. Scale bar: 50 μm n = 3 mice/group. ∗∗∗ p < 0.001 , ∗∗∗∗ p < 0.0001 , Student’s t test.

Article Snippet: The used primary antibodies included: rabbit anti-TLR2, 1:200, Abcam (ab209216); mouse Anti-His-tag, 1:200, MBL (D291-3); goat anti-IBA1, 1:400, Abcam (ab5076); Chicken anti-GFAP, 1:400, Abcam (ab4674); mouse anti-NeuN, 1:1000, Abcam (ab104224); mouse anti-c-Fos, 1:1000, Abcam (ab208942); rabbit anti-p-ERK, 1:400, CST (4370); rabbit anti-S2E, 1:500, Proteintech (28904); rabbit anti-S2S, 1:500, GeneTex (GTX635654); rabbit anti-S2M, 1:500, GeneTex (GTX636245); rabbit mAb isotype IgG, 1:500, CST(3900).

Techniques: Injection, Immunofluorescence

Journal: iScience

Article Title: TLR2/NF-κB signaling in macrophage/microglia mediated COVID-pain induced by SARS-CoV-2 envelope protein

doi: 10.1016/j.isci.2024.111027

Figure Lengend Snippet: The algesiogenic effects of SARS-CoV-2 viral proteins via i.t. injection (A–D) Paw withdrawal frequency to 0.4 g von Frey filament and withdrawal latency in Hargreaves test of male and female mice after i.t. injection of S2E with gradually increasing doses (0.01 μg, 0.1 μg and 1 μg). n = 6 mice/group. # or & p < 0.05 , && p < 0.01 , ∗∗∗ or ### or &&& p < 0.001 , ∗∗∗∗ or #### or &&&& p < 0.0001 vs. PBS, two-way ANOVA for repeated measures followed by Dunnett’s multiple comparisons test. (BL: Baseline). (E–H) Paw withdrawal frequency and latency in male and female mice after i.t. application of PBS, S2E, S2S, and S2M (1 μg). n = 6 mice/group; ∗ p < 0.05 , ∗∗ p < 0.01 , ∗∗∗∗ p < 0.0001 vs. PBS, two-way ANOVA for repeated measures followed by Dunnett’s post hoc test. (BL: Baseline). (I and J) Immunofluorescence of c-Fos (red) in SDH from mice receiving i.t. injection of PBS or S2E (blue for DAPI), along with quantitative analysis. Scale bar: 50 μm n = 3 mice/group. ∗∗∗∗ p < 0.0001 , Student’s t test.

Article Snippet: The used primary antibodies included: rabbit anti-TLR2, 1:200, Abcam (ab209216); mouse Anti-His-tag, 1:200, MBL (D291-3); goat anti-IBA1, 1:400, Abcam (ab5076); Chicken anti-GFAP, 1:400, Abcam (ab4674); mouse anti-NeuN, 1:1000, Abcam (ab104224); mouse anti-c-Fos, 1:1000, Abcam (ab208942); rabbit anti-p-ERK, 1:400, CST (4370); rabbit anti-S2E, 1:500, Proteintech (28904); rabbit anti-S2S, 1:500, GeneTex (GTX635654); rabbit anti-S2M, 1:500, GeneTex (GTX636245); rabbit mAb isotype IgG, 1:500, CST(3900).

Techniques: Injection, Immunofluorescence

Journal: iScience

Article Title: TLR2/NF-κB signaling in macrophage/microglia mediated COVID-pain induced by SARS-CoV-2 envelope protein

doi: 10.1016/j.isci.2024.111027

Figure Lengend Snippet:

Article Snippet: The used primary antibodies included: rabbit anti-TLR2, 1:200, Abcam (ab209216); mouse Anti-His-tag, 1:200, MBL (D291-3); goat anti-IBA1, 1:400, Abcam (ab5076); Chicken anti-GFAP, 1:400, Abcam (ab4674); mouse anti-NeuN, 1:1000, Abcam (ab104224); mouse anti-c-Fos, 1:1000, Abcam (ab208942); rabbit anti-p-ERK, 1:400, CST (4370); rabbit anti-S2E, 1:500, Proteintech (28904); rabbit anti-S2S, 1:500, GeneTex (GTX635654); rabbit anti-S2M, 1:500, GeneTex (GTX636245); rabbit mAb isotype IgG, 1:500, CST(3900).

Techniques: Recombinant, Protein Extraction, Endotoxin Assay, Western Blot, Virus, Software, Real-time Polymerase Chain Reaction, Imaging

Journal: Cell reports

Article Title: Uncovering the RNA-binding protein landscape in the pluripotency network of human embryonic stem cells.

doi: 10.1016/j.celrep.2021.109198

Figure Lengend Snippet: Figure 2. The STAT3 TF interacts directly with RNA in hESCs (A) Membrane image of infrared-labeled RNA in complex with STAT3. Protein-RNA complexes are visible as a smear (lanes 3 and 4) above the predicted mo- lecular weight of the STAT3-RNA-adaptor complex (marked with arrows). The lower arrow indicates the monomer and the upper arrow indicates the dimer form of STAT3, super-shifted due to bound RNA. The observed smear results from partial digestion with RNase, generating STAT3-RNA complexes of various sizes. At the higher RNase concentration (lane 3), the smear intensity is decreased due to increased RNA fragmentation. For clarity, gel lanes were cut at the indicated positions (white vertical lines). Lane 1: a non-CL control immunoprecipitated with an anti-STAT3 antibody. Lane 2: a mock IgG control. Lane 3: a CL STAT3-IP sample treated with a high concentration of RNase (1:100 dilution). Lane 4: same as lane 3, but with a lower concentration of RNase (1:500 dilution). (B) Results from seCLIP-seq using two biological replicates of STAT3 CLIP, a size-matched input (SMInput), and a non-CL sample. Magenta dots represent enriched peaks defined as those with more than 2-fold enrichment in CLIP over control (SMInput + non-CL) and probability above 0.8. Only peaks with fold enrichment > 1 are shown. (C) Read coverage tracks around enriched STAT3 peaks along the NORAD, SERF2, and MAD2L2 genes. Enriched peaks are portrayed by colored boxes. CPM, counts per million. (D) CLIP-qPCR validation of STAT3 binding to NORAD. Results are presented as enrichment relative to input in CL and non-CL experiments. Samples were normalized to GAPDH.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-STAT3 (Figures 2A, S2A, and S2E, top panel) Cell Signaling Technology Cat# 12640; RRID:AB_2629499 Rabbit anti-SRSF1 (related to Figure S3) Bethyl Cat# A302-052A; RRID:AB_1604258 Rabbit anti-OCT4 (Figures S8A, right panel, and S10A) Abcam Cat# ab19857; RRID:AB_445175 Rabbit anti-LIN28A (Figures S1B and S6G) Abcam Cat# ab124765; RRID:AB_10975201 Rabbit anti-ESRP1 (Figure S6G) Sigma Cat# HPA023719; RRID:AB_1856124 Rabbit anti-RBM47 (Figure S6G) Abcam Cat# ab167164; RRID:AB_2861348 Rabbit anti-RBPMS2 (Figure S6G) Abcam Cat# ab181098; RRID:AB_2861349 Rabbit anti-SOX2 (Figure S8A, right panel) Abcam Cat# ab97959; RRID:AB_2341193 Rabbit anti-NANOG (Figure S8A, right panel) Abcam Cat# ab109250; RRID:AB_10863442 Mouse anti-GAPDH (Figures S2E, top panel, S6G, and S8A, right panel) Abcam Cat# ab8245; RRID:AB_2107448 Mouse anti-hnRNPC1+C2 (Figure S1B) Abcam Cat# ab10294; RRID:AB_297034 Rabbit anti-Histone H3 (Figure S1B) Abcam Cat# ab1791; RRID:AB_302613 Chemicals, peptides, and recombinant proteins Recombinant human bFGF R&D Systems Cat# 233-FB-001MG/CF mTeSR1 complete kit Stem Cell Technologies Cat# 85850 Matrigel matrix Corning Cat# 354234 DMEM/F12 Biological Industries Cat# 01-170-1A KnockOut serum replacement GIBCO Cat# 10828-028 L-glutamine Biological Industries Cat# 03-020-1B 2-mercaptoethanol GIBCO Cat# 31350-010 MEM non-essential amino acids GIBCO Cat# 11140-035 Penicillin-streptomycin Biological Industries Cat# 03-031-1B Y-27632 Cayman Chemical Cat# 10005583 DMEM, high glucose Biological Industries Cat# 01-052-1A Fetal bovine serum Biological Industries Cat# 04-007-1A Certified fetal bovine serum (qualified for hESCs) Biological Industries Cat# 04-002-1A Opti-MEM I reduced serum medium GIBCO Cat# 31985062 Ca-Mg- PBS Biological Industries Cat# 02-023-1A Tris-HCl Sigma-Aldrich Cat# T2319 IGEPAL CA-630 Sigma-Aldrich Cat# I8896 NaCl Promega Cat# V4221 Sodium deoxycholate Sigma-Aldrich Cat# D6750 Lithium chloride solution Sigma-Aldrich Cat# L7026 Lithium dodecyl sulfate Sigma-Aldrich Cat# L9781 Sodium dodecyl sulfate solution Sigma-Aldrich Cat# 05030 TCEP Sigma-Aldrich Cat# 646547 PMSF Sigma-Aldrich Cat# 93482 EDTA Promega Cat# V4231 DL-Dithiothreitol solution Sigma-Aldrich Cat# 43816 StemPro accutase cell dissociation reagent GIBCO Cat# A1110501 (Continued on next page) Cell Reports 35, 109198, June 1, 2021 e1

Techniques: Membrane, Labeling, Concentration Assay, Control, Immunoprecipitation, Biomarker Discovery, Binding Assay