Journal: Frontiers in Immunology

Article Title: Nocardia rubra cell wall skeleton-induced MARCO expression: implications for improved phagocytosis and cytokine secretion in tumor-associated macrophages

doi: 10.3389/fimmu.2026.1611476

Figure Lengend Snippet: Nr-CWS affects MARCO expression through TLR4. The expression of TLR4 significantly increased after Nr-CWS treatment [ (D-G) , scale bars = 100 μm], while the expression TLR2 was unchanged (A-C) . Subsequent inhibition of TLR4 expression resulted in decreased levels of MARCO [ (H-J) , scale bars = 100 μm]. n = 3, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The following commercial antibodies (vendor, catalog number and dilution) utilized for western blot and immunohistochemical staining were employed in accordance with the manufacturers’ guidelines: mouse anti-CD68 antibody (Abcam, ab201340, 1:200), rabbit anti-MARCO antibody (Abcam, ab231046, 1:1000 for western blot and 1:100 for immunohistochemistry), rabbit anti-CD163 antibody (Abways Technology, CY6845, 1:500), mouse anti-CD86 antibody (Proteintech, 68674-2-Ig, 1:5000), mouse anti-TLR4 antibody (Santa Cruz Biotechnology, sc-293072, 1:1000 for western blot and 1:200 for immunohistochemistry), rabbit anti-TLR2 antibody(Abways Technology, CY5102, 1:2000), mouse anti-GAPDH antibody (Proteintech, 6004-1-Ig, 1:50000), HRP-goat anti-mouse recombinant secondary antibody (H+L)(Proteintech, RGAM001, 1:5000), goat anti-mouse IgG (H+L) Alexa Fluor 594 (Abways Technology, AB0152, 1:300), HRP-goat anti-rabbit recombinant secondary antibody (H+L) (Proteintech, RGAR001, 1:5000), goat anti-rabbit IgG (H+L) secondary antibody DyLightTM 594 (Report Biotech, S7002, 1:500), goat anti-mouse IgG (H+L) secondary antibody DyLightTM 488 (Report Biotech, S6001, 1:500), goat anti-mouse IgG (H+L) secondary antibody DyLightTM 594 (Abways Technology, AB0152, 1:500).

Techniques: Expressing, Inhibition

Journal: Virchows Archiv : an international journal of pathology

Article Title: Toll-like receptors 1, 2, 4, 5, and 6 in gastric cancer.

doi: 10.1007/s00428-023-03635-1

Figure Lengend Snippet: Fig. 1 Representative images of cytoplasmic TLR2 and TLR6 expression immunostaining in gastric adenocarcinoma. Low cytoplasmic TLR2 expression (A), high cytoplasmic TLR2 expression (B), low cytoplasmic TLR6 expression (C), and high cytoplasmic TLR6 expression (D)

Article Snippet: Samples were rinsed in distilled water and phosphate-buffered saline with Tween (PBS-T) and endogenous peroxidase was then neutralized in peroxidase blocking solution (Dako S2023) for 5 min. After a wash in PBS-T, sections were incubated with antibodies (Dako S2022); TLR1 (diluted 1:300, ROCKLAND 800-656-7625), TLR2 (diluted 1:500, ROCKLAND 600-401-956), TLR4 (diluted 1:1000, abonova 214-291), TLR5 (1:75, NOVUS NBP-2-24787), and TLR6 (diluted 1:750, abonova PAB 3555).

Techniques: Expressing, Immunostaining

Journal: PLOS Pathogens

Article Title: PRRSV-2 impedes lysosomes from eliminating secondary infected bacteria

doi: 10.1371/journal.ppat.1014000

Figure Lengend Snippet: (A) HEK-293T cells were co-transfected with the plasmids of RhoA and nsp5 for 24 h and then incubated with MG132, Z-VAD and 3-MA for 6 h, the cells were harvested to detect the RhoA and nsp5 protein expression by Western blot, tubulin served as an internal control. (B) HEK-293T cells were co-transfected with the designated plasmids for 36 h, the cells were harvested to detect the subcellular localization of RhoA and LC3. The co-localization of RhoA and LC3 was analyzed using ImageJ, and the results were quantified using the Pearson’s correlation coefficient. n = 10 cells from three independent experiments. (C) ATG7 +/+ and ATG7 -/- HEK-293T cells were co-transfected with the plasmids of RhoA and nsp5 for 36 h, the cells were harvested to detect the RhoA, nsp5 and ATG7 protein expression by Western blot, tubulin served as an internal control. (D) ATG7 -/- HEK-293T cells were co-transfected with the plasmids of RhoA, nsp5 and ATG7 for 36 h, the cells were harvested to detect the RhoA, nsp5 and ATG7 protein expression by Western blot, tubulin served as an internal control. (E) The plasmid of nsp5 was co-transfected with TAX1 BP1, NBR1, TOLLIP, PSMD4 and OPTN into HEK-293T cells for 36 h respectively, the cells were harvested to detect autophagy receptors that interact with nsp5 by Co-IP. (F) HEK-293T cells were co-transfected with the plasmids of nsp5 and TOLLIP for 36 h, the cells were harvested to detect the interaction between nsp5 and TOLLIP by Co-IP. (G) The plasmid of RhoA was co-transfected with TAX1 BP1, NBR1, TOLLIP, PSMD4 and OPTN into HEK-293T cells for 36 h respectively, the cells were harvested to detect autophagy receptors that interact with RhoA by Co-IP. (H) HEK-293T cells were co-transfected with the plasmids of RhoA and TOLLIP for 36 h, the cells were harvested to detect the interaction between RhoA and TOLLIP by Co-IP. (I) PAMs were infected with recombinant lentivirus expressing nsp5 or control lentivirus for 24 h, the cells were harvested to detect the interaction between nsp5 and endogenous RhoA, as well as endogenous TOLLIP by Co-IP. (J) HEK-293T cells were co-transfected with Flag-TOLLIP, Myc-RhoA, and HA-nsp5 plasmids for 36 h, the cells were harvested to detect the subcellular localization of TOLLIP, RhoA and nsp5. The co-localization of TOLLIP, RhoA and nsp5 was analyzed using ImageJ, and the results were quantified using the Pearson’s correlation coefficient. n = 10 cells from three independent experiments. (K) HEK-293T cells were co-transfected with HA-nsp5, Flag-TOLLIP and Myc-RhoA plasmids for 36 h, the cells were harvested to detect the interaction between nsp5, RhoA and TOLLIP by ternary Co-IP. (L) HEK-293T cells were co-transfected with the plasmids of RhoA, nsp5 and TOLLIP for 36 h, the cells were harvested to detect the RhoA, nsp5 and TOLLIP protein expression by Western blot, tubulin served as an internal control. (M) TOLLIP +/+ and TOLLIP -/- HEK-293T cells were co-transfected with the plasmids of RhoA and nsp5 for 36 h, the cells were harvested to detect the RhoA, nsp5 and TOLLIP protein expression by Western blot, tubulin served as an internal control. (N) TOLLIP -/- HEK-293T cells were co-transfected with the plasmids of RhoA, nsp5 and TOLLIP for 36 h, the cells were harvested to detect the RhoA, nsp5 and TOLLIP protein expression by Western blot, tubulin served as an internal control. (O) PAMs were transfected with siNC and siTOLLIP for 24 h, and then infected with recombinant lentivirus expressing nsp5 or control lentivirus for 24 h, the cells were harvested to detect the RhoA, nsp5 and TOLLIP protein expression by Western blot, tubulin served as an internal control. Scale bar: 10 μm. P values were calculated using Student’s t -test. ***, P < 0.001.

Article Snippet: Anti-RhoA monoclonal antibody (#2117) was purchased from Cell Signaling Technology, Anti-Smurf1 (sc-100616) was purchased from Santa Cruz Biotechnology, anti-RhoA antibody (10749–1-AP, 66733–1-Ig), anti-ATG7 polyclonal antibody (10088–2-AP), anti-TOLLIP polyclonal antibody (11315–1-AP), anti-LAMP1 monoclonal antibody (CL647–65051), anti-ubiquitin Polyclonal antibody (80992–1-RR) were purchased from Proteintech, anti-tubulin monoclonal antibody (HC-101) was purchased from TransGen Biotech, anti-Flag antibody (20543–1-AP, CL594–66008), anti-Myc antibody (16286–1-AP, CL488–60003), anti-GFP polyclonal antibody (50430–2-AP), anti-HA antibody (51064–2-AP, CL647–81290) were purchased from Proteintech, anti-PRRSV-2 N monoclonal antibody was prepared by our laboratory; Lipofectamine RNAiMAX Transfection Reagent (13778150), Lipofectamine 3000 Transfection Reagent (L3000015) and LysoTracker Red DND-99 (L7528) were purchased from Thermo Fisher Scientific; CFDA (S1076) was purchased from Solarbio; YF 488-Phalloidin (YP0114L) was purchased from UElandy; Latrunculin A (HY-16929), 3 × Flag peptide (HY-P0319) were purchased from MedChemExpress; ELISA assay kits for porcine IL-1β (SEKP-0001) and IL-6 (SEKP-0004) were procured from Solarbio.

Techniques: Transfection, Incubation, Expressing, Western Blot, Control, Plasmid Preparation, Co-Immunoprecipitation Assay, Infection, Recombinant