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Proteintech rabbit 10749 1 ap
Rabbit 10749 1 Ap, supplied by Proteintech, used in various techniques. Bioz Stars score: 96/100, based on 202 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/rabbit+anti+rhoa/RHOA+Antibody/pm41922321-225-18-34
Average 96 stars, based on 202 article reviews

rabbit 10749 1 ap - by Bioz Stars, 2026-09

96/100 stars

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Membrane:

Article Title: Chinese massage therapy (Tuina) inhibits motor neuron apoptosis in rats with sciatic nerve injury by regulating the cPLA2 and RhoA/ROCK2 signaling pathways
Article Snippet: .. The blocked membrane underwent overnight incubation at 4°C with the following primary antibodies: rabbit anti-GAPDH (1:3000, 10,494-1-AP, Proteintech Group, Inc., China), rabbit anti-RhoA (1:1000, 10,749-1-AP, Proteintech Group, Inc., China), rabbit anti-ROCK2 (1:1000, 21,645-1-AP, Proteintech Group, Inc., China), rabbit anti-Bax (1:1000, 60,267-1-Ig, Proteintech Group, Inc., China), rabbit anti-Bcl-2 (1:1000, 26,593-1-AP, Proteintech Group, Inc., China), rabbit anti-cPLA2 (1:1000, AF6329, Jiangsu Qinke Bio Research Center Co., Ltd., China), and rabbit anti-p-cPLA2 (1:1000, AF3329, Jiangsu Qinke Bio Research Center Co., Ltd., China). ..

Article Title: The NF-Y splicing signature controls hybrid EMT and ECM-related pathways to promote aggressiveness of colon cancer.
Article Snippet: .. Equivalent amounts of cellular extracts were resolved by SDS-PAGE, transferred to nitrocellulose membrane (GE Healthcare) with Trans-Blot Turbo Transfer System (Bio-Rad, USA) and immunoblotted with the following primary antibodies, diluted 1:1000 in 1X TBS with 1 mg/ml BSA: rabbit anti-vimentin (#D21H3 XP, Cell Signaling), rabbit anti-Snail (#C15D3, Cell Signaling), rabbit antiSlug (#C19G7, Cell Signalling), rabbit anti-E-cadherin (#3195, Cell Signaling), mouse anti–NF–YA (G2) (#sc-17753, Santa Cruz Biotechnology), rabbit anti-AKT (#MAB2055, R&D Systems), rabbit antiphospho-AKT(S473) (#AF887, R&D Systems), rabbit anti-RhoA (67B9, #9968, Cell Signaling),rabbit anti-RhoC (D40E4, #9968, Cell Signaling),mouse anti-Tubulin (#66031, Proteintech Europe). .. Membranes were blotted and scanned with Amersham Imager AI680 RGB (GE Healthcare), using chemiluminescent detection reagents Westar ηC and Supernova HRP substrates (Cyanagen).

Incubation:

Immunostaining:

Article Title: PIEZO1 regulates leader cell formation and cellular coordination during collective keratinocyte migration.
Article Snippet: .. Immunostaining was performed as previously described [84] using the following antibodies: Mouse antiRac1 (Millipore Cat#05–389-25UG, 1:200), Rabbit anti-RhoA (Proteintech Cat#10749–1-AP, 1:100), Donkey anti-Mouse 647 (Abcam Cat#AB150107, 1:500), Goat anti-Rabbit 488 (Life Sciences Cat#A32731, 1:500). .. Nuclei were stained by Hoechst (Invitrogen Cat#H1399) at 1μg/ mL for 5 minutes.

Article Title: PIEZO1 regulates leader cell formation and cellular coordination during collective keratinocyte migration
Article Snippet: .. Immunostaining was performed as previously described [ ] using the following antibodies: Mouse anti-Rac1 (Millipore Cat#05–389-25UG, 1:200), Rabbit anti-RhoA (Proteintech Cat#10749–1-AP, 1:100), Donkey anti-Mouse 647 (Abcam Cat#AB150107, 1:500), Goat anti-Rabbit 488 (Life Sciences Cat#A32731, 1:500). .. Nuclei were stained by Hoechst (Invitrogen Cat#H1399) at 1 μ g/mL for 5 minutes.

Image Search Results

(A) Immortalized PAMs were transfected with siNC and three distinct RhoA-specific siRNA for 36 h, the cells were harvested to detect the RhoA protein expression by Western blot, tubulin served as an internal control. (B) Immortalized PAMs were transfected with siNC or siRhoA for 36 h, the cells were harvested to observe the changes in F-actin. The fluorescence intensity of F-actin was quantified using ImageJ, n = 20 cells from three independent experiments. (C) Immortalized PAMs were transfected with siNC or siRhoA for 36 h and then infected with A. viridans for 4 h, the cells were harvested to detect the number of bacteria by CFUs. (D) Immortalized PAMs were transfected with siNC or siRhoA for 36 h and then infected with A. viridans for 4 h, the cells were harvested to detect the subcellular localization for lysosome and A. viridans. Colocalization between lysosome and A. viridans was quantified using ImageJ, with results expressed as the Manders’ correlation coefficients (M1 and M2). Specifically, M1 represents the proportion of lysosomal signal that overlaps with the bacterial signal relative to the total lysosomal signal, while M2 represents the proportion of bacterial signal overlapping with the lysosomal signal relative to the total bacterial signal. n = 20 cells from three independent experiments. (E) Immortalized PAMs were transfected with p3 × Flag (vector) or p3 × Flag-RhoA for 36 h, the cells were harvested to observe the changes in F-actin. The fluorescence intensity of F-actin was quantified using ImageJ, n = 20 cells from three independent experiments. (F) Immortalized PAMs were transfected with p3 × Flag (vector) or p3 × Flag-RhoA for 36 h and then infected with A. viridans for 4 h, the cells were harvested to detect the number of bacteria by CFUs. (G) Immortalized PAMs were transfected with p3 × Flag (vector) or p3 × Flag-RhoA for 36 h and then infected with A. viridans for the indicated periods, the cells were harvested to detect the subcellular localization for lysosome and A. viridans. Colocalization between lysosome and A. viridans was quantified using ImageJ, with results expressed as the Manders’ correlation coefficients (M1 and M2). Specifically, M1 represents the proportion of lysosomal signal that overlaps with the bacterial signal relative to the total lysosomal signal, while M2 represents the proportion of bacterial signal overlapping with the lysosomal signal relative to the total bacterial signal. n = 20 cells from three independent experiments. Scale bar: 10 μm. P values were calculated using Student’s t -test. ns, not significant; **, P < 0.01; ***, P < 0.001.

Journal: PLOS Pathogens

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

doi: 10.1371/journal.ppat.1014000

Figure Lengend Snippet: (A) Immortalized PAMs were transfected with siNC and three distinct RhoA-specific siRNA for 36 h, the cells were harvested to detect the RhoA protein expression by Western blot, tubulin served as an internal control. (B) Immortalized PAMs were transfected with siNC or siRhoA for 36 h, the cells were harvested to observe the changes in F-actin. The fluorescence intensity of F-actin was quantified using ImageJ, n = 20 cells from three independent experiments. (C) Immortalized PAMs were transfected with siNC or siRhoA for 36 h and then infected with A. viridans for 4 h, the cells were harvested to detect the number of bacteria by CFUs. (D) Immortalized PAMs were transfected with siNC or siRhoA for 36 h and then infected with A. viridans for 4 h, the cells were harvested to detect the subcellular localization for lysosome and A. viridans. Colocalization between lysosome and A. viridans was quantified using ImageJ, with results expressed as the Manders’ correlation coefficients (M1 and M2). Specifically, M1 represents the proportion of lysosomal signal that overlaps with the bacterial signal relative to the total lysosomal signal, while M2 represents the proportion of bacterial signal overlapping with the lysosomal signal relative to the total bacterial signal. n = 20 cells from three independent experiments. (E) Immortalized PAMs were transfected with p3 × Flag (vector) or p3 × Flag-RhoA for 36 h, the cells were harvested to observe the changes in F-actin. The fluorescence intensity of F-actin was quantified using ImageJ, n = 20 cells from three independent experiments. (F) Immortalized PAMs were transfected with p3 × Flag (vector) or p3 × Flag-RhoA for 36 h and then infected with A. viridans for 4 h, the cells were harvested to detect the number of bacteria by CFUs. (G) Immortalized PAMs were transfected with p3 × Flag (vector) or p3 × Flag-RhoA for 36 h and then infected with A. viridans for the indicated periods, the cells were harvested to detect the subcellular localization for lysosome and A. viridans. Colocalization between lysosome and A. viridans was quantified using ImageJ, with results expressed as the Manders’ correlation coefficients (M1 and M2). Specifically, M1 represents the proportion of lysosomal signal that overlaps with the bacterial signal relative to the total lysosomal signal, while M2 represents the proportion of bacterial signal overlapping with the lysosomal signal relative to the total bacterial signal. n = 20 cells from three independent experiments. Scale bar: 10 μm. P values were calculated using Student’s t -test. ns, not significant; **, P < 0.01; ***, 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, Expressing, Western Blot, Control, Fluorescence, Infection, Bacteria, Plasmid Preparation

(A-B) PAMs were infected with PRRSV-2 (MOI = 1) for the indicated periods, and the cells were harvested to detect (A) RhoA mRNA expression by RT-qPCR, (B) RhoA protein expression by Western blot, tubulin served as an internal control, and the PRRSV-2 N protein was used as an infection indicator. (C) HEK-293T cells were co-transfected with the plasmids of PRRSV-2 proteins and RhoA for 36 h, and the cells were harvested to detect the PRRSV-2 proteins and RhoA protein expression by Western blot, tubulin served as an internal control. (D) HEK-293T cells were co-transfected with the plasmids of RhoA and nsp5 with an increasing concentration gradient for 36 h, the cells were harvested to detect the RhoA and PRRSV-2 nsp5 protein expression by Western blot, tubulin served as an internal control. (E-F) HEK-293T cells were co-transfected with the plasmids of RhoA and nsp5 for 36 h, the cells were harvested to detect the interaction between RhoA and nsp5 by Co-IP. (G) HEK-293T cells were co-transfected with the plasmids of RhoA and nsp2 for 36 h, the cells were harvested to detect the interaction between RhoA and nsp2 by Co-IP. (H) HEK-293T cells were co-transfected with the designated plasmids for 36 h, the cells were harvested to detect the subcellular localization of nsp5 and nsp2 in relation to RhoA, respectively. The co-localization of nsp2 and nsp5 with RhoA was analyzed using ImageJ, and the results were quantified using the Pearson’s correlation coefficient. n = 10 cells from three independent experiments. (I) Online prediction of the transmembrane domain distribution of nsp5 polypeptide sequences. (J) The truncated plasmids were constructed based on the transmembrane domain distribution of nsp5. (K) HEK-293T cells were co-transfected with the plasmids of RhoA and the truncated plasmid of nsp5 for 36 h, the cells were harvested to detect the RhoA and the truncated nsp5 protein expression by Western blot, tubulin served as an internal control. (L) The plasmid of RhoA was co-transfected with nsp5 and nsp5-D5 into HEK-293T cells for 36 h respectively, the cells were harvested to detect the RhoA, nsp5 and nsp5-D5 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.

Journal: PLOS Pathogens

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

doi: 10.1371/journal.ppat.1014000

Figure Lengend Snippet: (A-B) PAMs were infected with PRRSV-2 (MOI = 1) for the indicated periods, and the cells were harvested to detect (A) RhoA mRNA expression by RT-qPCR, (B) RhoA protein expression by Western blot, tubulin served as an internal control, and the PRRSV-2 N protein was used as an infection indicator. (C) HEK-293T cells were co-transfected with the plasmids of PRRSV-2 proteins and RhoA for 36 h, and the cells were harvested to detect the PRRSV-2 proteins and RhoA protein expression by Western blot, tubulin served as an internal control. (D) HEK-293T cells were co-transfected with the plasmids of RhoA and nsp5 with an increasing concentration gradient for 36 h, the cells were harvested to detect the RhoA and PRRSV-2 nsp5 protein expression by Western blot, tubulin served as an internal control. (E-F) HEK-293T cells were co-transfected with the plasmids of RhoA and nsp5 for 36 h, the cells were harvested to detect the interaction between RhoA and nsp5 by Co-IP. (G) HEK-293T cells were co-transfected with the plasmids of RhoA and nsp2 for 36 h, the cells were harvested to detect the interaction between RhoA and nsp2 by Co-IP. (H) HEK-293T cells were co-transfected with the designated plasmids for 36 h, the cells were harvested to detect the subcellular localization of nsp5 and nsp2 in relation to RhoA, respectively. The co-localization of nsp2 and nsp5 with RhoA was analyzed using ImageJ, and the results were quantified using the Pearson’s correlation coefficient. n = 10 cells from three independent experiments. (I) Online prediction of the transmembrane domain distribution of nsp5 polypeptide sequences. (J) The truncated plasmids were constructed based on the transmembrane domain distribution of nsp5. (K) HEK-293T cells were co-transfected with the plasmids of RhoA and the truncated plasmid of nsp5 for 36 h, the cells were harvested to detect the RhoA and the truncated nsp5 protein expression by Western blot, tubulin served as an internal control. (L) The plasmid of RhoA was co-transfected with nsp5 and nsp5-D5 into HEK-293T cells for 36 h respectively, the cells were harvested to detect the RhoA, nsp5 and nsp5-D5 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.

Techniques: Infection, Expressing, Quantitative RT-PCR, Western Blot, Control, Transfection, Concentration Assay, Co-Immunoprecipitation Assay, Construct, Plasmid Preparation

(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.

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.

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

(A) HEK-293T cells were co-transfected with the plasmids of RhoA, nsp5 and Ub for 36 h, the cells were harvested to detect the ubiquitination of RhoA induced by nsp5. (B) PAMs were infected with recombinant lentivirus expressing nsp5 or control lentivirus for 24 h, the cells were harvested to detect the ubiquitination of endogenous RhoA induced by nsp5. (C) 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 LAMP1. The co-localization of RhoA and LAMP1 was analyzed using ImageJ, and the results were quantified using the Pearson’s correlation coefficient. n = 10 cells from three independent experiments. (D) Seven types of ubiquitin plasmids (K6-Ub, K11-Ub, K27-Ub, K29-Ub, K33-Ub, K48-Ub, and K63-Ub) were co-transfected in HEK-293T cells with nsp5 and RhoA respectively for 36 h, the cells were harvested to detect the ubiquitination type of RhoA induced by nsp5. (E-H) The indicated lysine residue mutant plasmids of RhoA were co-transfected in HEK-293T cells with nsp5 for 36 h, the cells were harvested to detect the RhoA and nsp5 protein expression by Western blot, tubulin served as an internal control. (I) HEK-293T cells were co-transfected with the plasmids of RhoA K187R , nsp5 and Ub for 36 h, the cells were harvested to detect the ubiquitination of RhoA K187R induced by nsp5. (J) Immortalized PAMs were transfected with siNC and siRhoA for 24 h, and then transfected with the plasmids of RhoA K187R for 24 h, the cells were harvested to detect the RhoA protein expression by Western blot, tubulin served as an internal control. (K) Immortalized PAMs were transfected with siNC and siRhoA for 24 h, and then transfected with the plasmids of RhoA K187R for 24 h. Subsequently, cells were infected with A. viridans for 4 h, the cells were harvested to detect the number of bacteria by CFUs. (L) Immortalized PAMs were transfected with siNC and siRhoA for 24 h, and then transfected with the plasmids of RhoA K187R for 24 h. Subsequently, cells were infected with A. viridans for 4 h, the cells were harvested to detect the subcellular localization for lysosome and A. viridans . Colocalization between lysosome and A. viridans was quantified using ImageJ, with results expressed as the Manders’ correlation coefficients (M1 and M2). Specifically, M1 represents the proportion of lysosomal signal that overlaps with the bacterial signal relative to the total lysosomal signal, while M2 represents the proportion of bacterial signal overlapping with the lysosomal signal relative to the total bacterial signal. n = 20 cells from three independent experiments. Scale bar: 10 μm. P values were calculated using Student’s t -test. **, P < 0.01, ***, P < 0.001.

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, nsp5 and Ub for 36 h, the cells were harvested to detect the ubiquitination of RhoA induced by nsp5. (B) PAMs were infected with recombinant lentivirus expressing nsp5 or control lentivirus for 24 h, the cells were harvested to detect the ubiquitination of endogenous RhoA induced by nsp5. (C) 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 LAMP1. The co-localization of RhoA and LAMP1 was analyzed using ImageJ, and the results were quantified using the Pearson’s correlation coefficient. n = 10 cells from three independent experiments. (D) Seven types of ubiquitin plasmids (K6-Ub, K11-Ub, K27-Ub, K29-Ub, K33-Ub, K48-Ub, and K63-Ub) were co-transfected in HEK-293T cells with nsp5 and RhoA respectively for 36 h, the cells were harvested to detect the ubiquitination type of RhoA induced by nsp5. (E-H) The indicated lysine residue mutant plasmids of RhoA were co-transfected in HEK-293T cells with nsp5 for 36 h, the cells were harvested to detect the RhoA and nsp5 protein expression by Western blot, tubulin served as an internal control. (I) HEK-293T cells were co-transfected with the plasmids of RhoA K187R , nsp5 and Ub for 36 h, the cells were harvested to detect the ubiquitination of RhoA K187R induced by nsp5. (J) Immortalized PAMs were transfected with siNC and siRhoA for 24 h, and then transfected with the plasmids of RhoA K187R for 24 h, the cells were harvested to detect the RhoA protein expression by Western blot, tubulin served as an internal control. (K) Immortalized PAMs were transfected with siNC and siRhoA for 24 h, and then transfected with the plasmids of RhoA K187R for 24 h. Subsequently, cells were infected with A. viridans for 4 h, the cells were harvested to detect the number of bacteria by CFUs. (L) Immortalized PAMs were transfected with siNC and siRhoA for 24 h, and then transfected with the plasmids of RhoA K187R for 24 h. Subsequently, cells were infected with A. viridans for 4 h, the cells were harvested to detect the subcellular localization for lysosome and A. viridans . Colocalization between lysosome and A. viridans was quantified using ImageJ, with results expressed as the Manders’ correlation coefficients (M1 and M2). Specifically, M1 represents the proportion of lysosomal signal that overlaps with the bacterial signal relative to the total lysosomal signal, while M2 represents the proportion of bacterial signal overlapping with the lysosomal signal relative to the total bacterial signal. n = 20 cells from three independent experiments. Scale bar: 10 μm. P values were calculated using Student’s t -test. **, P < 0.01, ***, P < 0.001.

Techniques: Transfection, Ubiquitin Proteomics, Infection, Recombinant, Expressing, Control, Residue, Mutagenesis, Western Blot, Bacteria

(A) The plasmid of nsp5 was co-transfected with Smurf1, Smurf2, Cul3 and RhoA into HEK-293T cells for 36 h respectively, the cells were harvested to detect E3 ubiquitin ligase that interact with nsp5 by Co-IP. (B) The plasmid of Smurf1 was co-transfected with nsp5, RhoA and nsp2 into HEK-293T cells for 36 h respectively, the cells were harvested to detect nsp5 and RhoA that interact with Smurf1 by Co-IP. (C) 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 Smurf1 by Co-IP. (D) HEK-293T cells were co-transfected with Flag-Smurf1, Myc-RhoA, and HA-nsp5 plasmids for 36 h, the cells were harvested to detect the subcellular localization of Smurf1, RhoA and nsp5. The co-localization of Smurf1, 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. (E) HEK-293T cells were co-transfected with HA-nsp5, Flag- Smurf1 and Myc-RhoA plasmids for 36 h, the cells were harvested to detect the interaction between nsp5, RhoA and Smurf1 by ternary Co-IP. (F) HEK-293T cells were co-transfected with the plasmids of RhoA, Smurf1 and Ub for 36 h, the cells were harvested to detect the ubiquitination of RhoA induced by Smurf1. (G) HEK-293T cells were co-transfected with the plasmids of RhoA, Smurf1, nsp5 and Ub for 36 h, the cells were harvested to detect the effect of nsp5 on Smurf1-induced polyubiquitination of RhoA. (H) HEK-293T cells were co-transfected with the plasmids of RhoA, Smurf1, nsp5 and K63-Ub for 36 h, the cells were harvested to detect the effect of nsp5 on Smurf1-induced polyubiquitination of RhoA. (I) HEK-293T cells were co-transfected with the plasmids of RhoA, nsp5 and Smurf1 for 36 h, the cells were harvested to detect the RhoA, nsp5 and Smurf1 protein expression by Western blot, tubulin served as an internal control. (J) Smurf1 +/+ and Smurf1 -/- 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 Smurf1 protein expression by Western blot, tubulin served as an internal control. (K) Smurf1 -/- HEK-293T cells were co-transfected with the plasmids of RhoA, nsp5 and Smurf1 for 36 h, the cells were harvested to detect the RhoA, nsp5 and Smurf1 protein expression by Western blot, tubulin served as an internal control. (L) PAMs were transfected with siNC and siSmurf1 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 Smurf1 protein expression by Western blot, tubulin served as an internal control. Scale bar: 10 μm.

Journal: PLOS Pathogens

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

doi: 10.1371/journal.ppat.1014000

Figure Lengend Snippet: (A) The plasmid of nsp5 was co-transfected with Smurf1, Smurf2, Cul3 and RhoA into HEK-293T cells for 36 h respectively, the cells were harvested to detect E3 ubiquitin ligase that interact with nsp5 by Co-IP. (B) The plasmid of Smurf1 was co-transfected with nsp5, RhoA and nsp2 into HEK-293T cells for 36 h respectively, the cells were harvested to detect nsp5 and RhoA that interact with Smurf1 by Co-IP. (C) 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 Smurf1 by Co-IP. (D) HEK-293T cells were co-transfected with Flag-Smurf1, Myc-RhoA, and HA-nsp5 plasmids for 36 h, the cells were harvested to detect the subcellular localization of Smurf1, RhoA and nsp5. The co-localization of Smurf1, 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. (E) HEK-293T cells were co-transfected with HA-nsp5, Flag- Smurf1 and Myc-RhoA plasmids for 36 h, the cells were harvested to detect the interaction between nsp5, RhoA and Smurf1 by ternary Co-IP. (F) HEK-293T cells were co-transfected with the plasmids of RhoA, Smurf1 and Ub for 36 h, the cells were harvested to detect the ubiquitination of RhoA induced by Smurf1. (G) HEK-293T cells were co-transfected with the plasmids of RhoA, Smurf1, nsp5 and Ub for 36 h, the cells were harvested to detect the effect of nsp5 on Smurf1-induced polyubiquitination of RhoA. (H) HEK-293T cells were co-transfected with the plasmids of RhoA, Smurf1, nsp5 and K63-Ub for 36 h, the cells were harvested to detect the effect of nsp5 on Smurf1-induced polyubiquitination of RhoA. (I) HEK-293T cells were co-transfected with the plasmids of RhoA, nsp5 and Smurf1 for 36 h, the cells were harvested to detect the RhoA, nsp5 and Smurf1 protein expression by Western blot, tubulin served as an internal control. (J) Smurf1 +/+ and Smurf1 -/- 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 Smurf1 protein expression by Western blot, tubulin served as an internal control. (K) Smurf1 -/- HEK-293T cells were co-transfected with the plasmids of RhoA, nsp5 and Smurf1 for 36 h, the cells were harvested to detect the RhoA, nsp5 and Smurf1 protein expression by Western blot, tubulin served as an internal control. (L) PAMs were transfected with siNC and siSmurf1 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 Smurf1 protein expression by Western blot, tubulin served as an internal control. Scale bar: 10 μm.

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

(A-D) Immortalized PAMs were transfected with siNC or siRhoA for 24 h and then infected with PRRSV-2 for 36 h, cells and supernatants were harvested to determine (A) RhoA mRNA expression, (B) PRRSV-2 ORF7 mRNA expression, (C) RhoA and PRRSV-2 N protein expression, tubulin served as an internal control, (D) supernatant virus titer. (E-F) Immortalized PAMs were transfected with pEGFP-C1 (vector) or pEGFP-C1-RhoA for 24 h, followed by infection with PRRSV-2 for the indicated periods, cells and supernatants were harvested to determine (E) RhoA and PRRSV-2 N protein expression, tubulin served as an internal control, (F) supernatant virus titer. P values were calculated using Student’s t -test. ns, not significant.

Journal: PLOS Pathogens

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

doi: 10.1371/journal.ppat.1014000

Figure Lengend Snippet: (A-D) Immortalized PAMs were transfected with siNC or siRhoA for 24 h and then infected with PRRSV-2 for 36 h, cells and supernatants were harvested to determine (A) RhoA mRNA expression, (B) PRRSV-2 ORF7 mRNA expression, (C) RhoA and PRRSV-2 N protein expression, tubulin served as an internal control, (D) supernatant virus titer. (E-F) Immortalized PAMs were transfected with pEGFP-C1 (vector) or pEGFP-C1-RhoA for 24 h, followed by infection with PRRSV-2 for the indicated periods, cells and supernatants were harvested to determine (E) RhoA and PRRSV-2 N protein expression, tubulin served as an internal control, (F) supernatant virus titer. P values were calculated using Student’s t -test. ns, not significant.

Techniques: Transfection, Infection, Expressing, Control, Virus, Plasmid Preparation

During PRRSV-2 infection, PRRSV-2 nsp5 interacts with RhoA, inducing the autophagy-lysosome degradation of RhoA and resulting in a decrease in F-actin. The reduction of F-actin impedes the efficiency of lysosomal targeted clearance of bacteria, thereby facilitating the survival of secondary infected bacteria.

Journal: PLOS Pathogens

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

doi: 10.1371/journal.ppat.1014000

Figure Lengend Snippet: During PRRSV-2 infection, PRRSV-2 nsp5 interacts with RhoA, inducing the autophagy-lysosome degradation of RhoA and resulting in a decrease in F-actin. The reduction of F-actin impedes the efficiency of lysosomal targeted clearance of bacteria, thereby facilitating the survival of secondary infected bacteria.

Techniques: Infection, Bacteria

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