Journal: iScience

Article Title: Ubiquitination and degradation of CD47 enhances macrophage phagocytosis of hemolytic erythrocytes

doi: 10.1016/j.isci.2025.114499

Figure Lengend Snippet: Changes in the major protein components of the proteasome during hemolysis (A) Western blot analysis of proteasomal subunits (PSME1/2, PSMB5/6/7) expression in control and hemolysis groups. (B and C) Quantification of proteasomal subunits (PSME1/2, PSMB5/6/7) expression in the RBC membrane (B) and cytoplasm (C) ( n = 3). (D) Representative immunofluorescence images of proteasomal subunits (PSME1/2, PSMB5/6/7) of hemolytic RBCs ( n = 6). Scale bars = 10 μm. (E) Schematic illustration of the animal model of immune hemolysis. (F and G) Immunofluorescence of proteasomal subunits (PSME1/2, PSMB5/6/7) in hemolytic mouse RBCs (F) and AIHA patient RBCs (G) ( n = 6). Scale bars = 10 μm. (H–J) Statistical analysis of caspase-like activity (H), trypsin-like activity (I), and chymotrypsin-like activity (J) of the membrane proteins after hemolysis ( n = 6). Data were analyzed by Student’s t test (two groups) or one-way ANOVA with Tukey’s test (multiple groups) and are expressed as mean ± SEM. ∗ p < 0.05, ∗∗∗ p < 0.001, ns = no significance.

Article Snippet: The primary antibodies utilized in this procedure were CD47 (Santa Cruz Biotechnology, Cat# sc-12730), MARCH1 (HUABIO Biotechnology, Cat# ER63906), PSME1 (Abcam, Cat# ab186832), PSME2 (Abcam, Cat# ab183727), PSMB5 (BOSTER, Cat# A03418-1), PSMB6 (ABclonal Technology, Cat# A4053), PSMB7 (BOSTER, Cat# A08095-1), and UBQLN1 (Proteintech Group, Cat# 22126-1-AP).

Techniques: Western Blot, Expressing, Control, Membrane, Immunofluorescence, Animal Model, Activity Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2.

doi: 10.4049/jimmunol.181.4.2664

Figure Lengend Snippet: FIGURE 2. Effect of Rac1 siRNA and the Rac1 inhibitor NSC23766 on NOD2- and TLR2-mediated IL-8 secretion. Primary human monocytes were transfected with control nonsilencing siRNA (c-siRNA) or siRNA targeting Rac1 (si-Rac1). After 72 h, cells were lysed and Western blots using anti-Rac1 Abs were performed (A). Membranes were simultaneously probed with anti-ERK2 Abs to confirm equal protein loading. B, Primary human monocytes were transfected with siRNAs as indicated, and after 72 h, stimulated with MDP or Malp2 for 16 h, and the supernatants were analyzed for IL-8 secretion by ELISA. THP-1 cells were untreated (none) or preincubated overnight with the Rac1 inhibitor NSC23766 (200 M). Subsequently, the cells were either stimulated with MDP (C) or Malp2 (D) for 16 h, and the supernatants were analyzed for IL-8 secretion by ELISA. Data presented are mean SD of three different experiments performed in duplicates (, p 0.01).

Article Snippet: Activated Rac1 in the cell lysates was visualized by Western blot using an anti-Rac1 mAb and a Cy5.5-labeled anti-mouse secondary Ab (Rockland).

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

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2.

doi: 10.4049/jimmunol.181.4.2664

Figure Lengend Snippet: FIGURE 1. Activation of Rac1 by the NOD2 agonist MDP in THP-1 cells and primary human monocytes. THP-1 cells (A) were stimulated with either the NOD2 agonist MDP-LD (10 g/ ml) or with the inactive compound MDP-DD (10 g/ml), or primary human monocytes (B) were treated with MDP-LD for the indicated time intervals, and Rac1 activation was deter- mined by the amount bound to the GST-PAK Rac1 interaction binding site (PD, pulldown). C, THP-1 cells were stimulated with MDP-LD in concentrations as indicated for 30 min and Rac1 activation was determined by Rac1 pulldown as- say. Activated GTPase levels were normalized to the amount of total Rac1 in cell lysates (IB, immunoblot) as analyzed by Western blotting. The intensity of the bands was quantified and the values are the mean SD (error bars) of three independent experiments normalized to the re- sponse of untreated cells.

Article Snippet: Activated Rac1 in the cell lysates was visualized by Western blot using an anti-Rac1 mAb and a Cy5.5-labeled anti-mouse secondary Ab (Rockland).

Techniques: Activation Assay, Binding Assay, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2.

doi: 10.4049/jimmunol.181.4.2664

Figure Lengend Snippet: FIGURE 3. Influence of Rac1 on NOD2- or TLR2-mediated IL-8 and NF-B activation. A, HEK293 cells were left untreated, or were trans- fected with control non-silencing siRNA (c-siRNA) or siRNA targeting Rac1 (si-Rac1). After 72 h, cells were lysed and Western blots using anti-Rac1 Abs were performed in duplicates. Membranes were simul- taneously probed with anti-ERK2 Abs to confirm equal protein loading. B–D, HEK293 cells were left untreated (ctrl), or were transfected with control non-silencing siRNA (c-siRNA) or siRNA targeting Rac1 (si- Rac1). After 48 h, the cells were additionally cotransfected with NOD2 (B and C) or TLR2 (D) expression plasmids, together with an IL-8- reporter (B) or NF-B reporter construct (C and D) and a -galactosi- dase reporter plasmid. Cells were either left untreated () or stimulated with MDP (B and C) or Malp2 (D) and relative luciferase activities were obtained. E and F, HEK293 cells seeded in 24-well plates were tran- siently transfected with a control vector (ctrl) or NOD2 expression plas- mid along with an IL-8 luciferase reporter plasmid (E) or a NF-B- driven luciferase reporter (F), respectively, and a -galactosidase reporter plasmid. Additionally, the cells were cotransfected with wild- type Rac1 (Rac1wt), dominant negative Rac1N17 or constitutively ac- tive Rac1L61. Cells were either stimulated with 10 g/ml MDP (MDP) or left untreated (), and relative luciferase activities were obtained the next day. G, HEK293 cells seeded in 24-well plates were transiently transfected with a control vector (ctrl), or a RIP2 expression plasmid along with a NF-B-driven luciferase reporter and a -galactosidase plasmid. The influence of Rac1 was tested by additionally introducing wild-type Rac1 (Rac1wt), dominant negative Rac1 (Rac1N17), or con- stitutive active Rac1 (Rac1L61) mean SD; , p 0.01.

Article Snippet: Activated Rac1 in the cell lysates was visualized by Western blot using an anti-Rac1 mAb and a Cy5.5-labeled anti-mouse secondary Ab (Rockland).

Techniques: Activation Assay, Control, Western Blot, Transfection, Expressing, Construct, Plasmid Preparation, Luciferase, Dominant Negative Mutation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2.

doi: 10.4049/jimmunol.181.4.2664

Figure Lengend Snippet: FIGURE 4. Rac1 and NOD2 colocalized and interacted at the plasma membrane. A, Immunofluorescence micrographs of HEK293 cells cotrans- fected with NOD2 and Rac1 expression plasmids. Fixed cells were stained with anti-NOD2 and anti-Rac1 Abs. Signals obtained with the two Abs (left and middle panels) are presented and a merged image is shown in the right panel. B, Western blot analysis of coimmunoprecipitations from HEK293 cells are shown. Myc-NOD2 and Rac1 wild-type were ectopically ex- pressed in HEK293 cells and lysates were precipitated using anti-c-myc Ab (IP) and coprecipitated Rac1 was detected using anti-Rac1 Ab (IB). Vice versa, lysates were precipitated with anti-Rac1 (IP) and coprecipitated myc-NOD2 was detected using an anti-c-myc Ab (IB). MDP-stimulated THP-1 cells (C) or primary monocytes (D) were lysed at different time points, immunoprecipitations of endogenous NOD2 or endogenous Rac1 with the respective Abs were performed, and immune complexes were probed for the presence of Rac1 or NOD2, respectively. Equal loading was confirmed by blotting whole cell lysates with anti-Rac1 Ab (Input). E, MDP-stimulated THP-1 cells were immunoprecipitated with Abs directed against Nalp3 and immune complexes were probed for the presence of Rac1 or Nalp3. All experiments were repeated three times.

Article Snippet: Activated Rac1 in the cell lysates was visualized by Western blot using an anti-Rac1 mAb and a Cy5.5-labeled anti-mouse secondary Ab (Rockland).

Techniques: Clinical Proteomics, Membrane, Expressing, Staining, Western Blot, Immunoprecipitation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2.

doi: 10.4049/jimmunol.181.4.2664

Figure Lengend Snippet: FIGURE 5. Influence of Rac1 inhibition on recruit- ment of NOD2 and Rac1 to the plasma membrane. A, THP-1 cells were left untreated or preincubated with the Rac1 inhibitor NSC23766 (NSC) overnight. The next day, the cells were left untreated () or stimulated with 10 g/ml MDP (MDP) for 40 min. Membrane fractions were separated and immunoblotted with anti-NOD2 or anti-Rac1 Abs. The intensity of the bands was quanti- fied and the values are the mean SD (error bars) of three independent experiments normalized to untreated cells and indicated as fold activation. B, Primary human monocytes were mock-transfected (none), transfected with control non-silencing siRNA (c-siRNA) or siRNA targeting Rac1 (si-Rac1) and, after 72 h, stimulated with MDP for 40 min. Membrane and cytosol fractions were separated and immunoblotted with anti-NOD2 Abs. All experiments were repeated three times.

Article Snippet: Activated Rac1 in the cell lysates was visualized by Western blot using an anti-Rac1 mAb and a Cy5.5-labeled anti-mouse secondary Ab (Rockland).

Techniques: Inhibition, Clinical Proteomics, Membrane, Activation Assay, Transfection, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2.

doi: 10.4049/jimmunol.181.4.2664

Figure Lengend Snippet: FIGURE 7. Rac1 and -PIX siRNAs as well as the Rac1 inhibitor NSC23766 inhibit interaction of NOD2 with Erbin. Primary monocytes (A and B) or THP-1 cells (C) were either preincubated with the Rac1 inhibitor NSC23766 (NSC) or were transfected with Rac1 siRNA or -PIX siRNA, as indicated, and were stimulated with 10 g/ml MDP (MDP) for 40 min. Subsequently, immunoprecipitations with an Erbin Ab and subsequent im- munoblots with NOD2 and ERK2 Abs (A) or NOD2, Rac1, and Erbin Abs (B and C) were performed. One representative Western blot out of three is shown.

Article Snippet: Activated Rac1 in the cell lysates was visualized by Western blot using an anti-Rac1 mAb and a Cy5.5-labeled anti-mouse secondary Ab (Rockland).

Techniques: Transfection, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2.

doi: 10.4049/jimmunol.181.4.2664

Figure Lengend Snippet: FIGURE 6. Involvement of -PIX in NOD2-medi- ated signaling. MDP-stimulated primary monocytes or THP-1 cells (A and B) were lysed at different time points, immunoprecipitations of endogenous Rac1 (A) or endogenous NOD2 (B) with the respective Abs were performed, and immune complexes were probed for the presence of -Pix. Equal protein amounts in the lysates were confirmed by blotting total cell lysates with an ERK2 Ab (Input). All experiments were repeated three times. C, THP-1 cells were transfected with control non-silencing siRNA (c-siRNA) or siRNA targeting -Pix (si--Pix_S1 (sequence 1), si-Pix_S2 (sequence 2)). After 72 h, cells were lysed and Western blots using anti--Pix Abs were performed. Western blots were si- multaneously probed with anti-ERK2 Abs to confirm equal protein loading. D, THP-1 cells were transfected as indicated, incubated for 72 h, and stimulated with MDP (10 g/ml) for 40 min. Membrane and cytosol fractions were separated and immunoblotted with anti- NOD2 Abs. E and F, THP-1 cells were transfected with siRNAs as indicated, and after 72 h, stimulated with MDP (E) or Malp2 (F) for 16 h, and the supernatants were analyzed for IL-8 secretion by ELISA. Data pre- sented are mean SD of three different experiments performed in duplicates (, p 0.01).

Article Snippet: Activated Rac1 in the cell lysates was visualized by Western blot using an anti-Rac1 mAb and a Cy5.5-labeled anti-mouse secondary Ab (Rockland).

Techniques: Transfection, Control, Sequencing, Western Blot, Incubation, Membrane, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2.

doi: 10.4049/jimmunol.181.4.2664

Figure Lengend Snippet: FIGURE 8. Schema of the molecular association among NOD2, -PIX, Rac1, and Erbin as discussed in the text.

Article Snippet: Activated Rac1 in the cell lysates was visualized by Western blot using an anti-Rac1 mAb and a Cy5.5-labeled anti-mouse secondary Ab (Rockland).

Techniques: