Journal: Frontiers in Immunology

Article Title: Establishment and characterization of an immortalized red river hog blood-derived macrophage cell line

doi: 10.3389/fimmu.2024.1465952

Figure Lengend Snippet: LPS-induced increases in the expression of inflammatory cytokine and interferon β mRNAs in RZJ/IBM cells. Total RNA was recovered from RZJ/IBM cells treated with or without 1 μg/mL LPS, and RNA-seq experiments were performed independently three times. The transcripts per million (TPM) values of the genes indicated were expressed as mean ± SEM values. The TPM value of GAPDH was used as an internal control.

Article Snippet: The primary antibodies used for immunoblotting were as follows: rabbit monoclonal antibodies against phospho-p38 MAPK (D3F9, Cat. No. #4511), p38 MAPK (D13E1, Cat. No. #8690), and phospho-NF-κB p65 (93H1, Cat. No. #3033) (Cell Signaling Technology, Inc., Danvers, MA); rabbit polyclonal antibodies against NF-κB p65 (Cat. No. #3034, Cell Signaling Technology); biotinylated antibodies against IL-1β (Cat. No. BAF681) and IL-18 (Cat. No. BAF588) (R&D Systems, Inc., Minneapolis, MN); and mouse monoclonal antibodies against GAPDH (MAb 6C5) (Cat. No. 5G4, HyTest Ltd., Turku, Finland).

Techniques: Expressing, RNA Sequencing, Control

Journal: Frontiers in Immunology

Article Title: Establishment and characterization of an immortalized red river hog blood-derived macrophage cell line

doi: 10.3389/fimmu.2024.1465952

Figure Lengend Snippet: Phosphorylation of p65 NF-κB and p38 MAPK, and the production of IL-1β and IL-18 in RZJ/IBM cells in response to LPS or MDP. The treatment with LPS or MDP induced the phosphorylation of NF-κB p65 and p38 MAPK in a dose-dependent manner ( A , first and third panels ). The equivalent protein loading of these molecules was confirmed by immunoblotting with anti-NF-κB p65 or anti-p38 MAPK antibodies ( A , second and fourth panels ). The dose-dependent production of pro-IL-1β and pro-IL-18 was also detected in cell lysates ( B , first and third panels ) or culture supernatants ( B , second and fourth panels ) from RZJ/IBM cells that had been stimulated with LPS for 3 days. The secretion of mIL-18 from LPS-treated RZJ/IBM cells into the culture supernatant was also detected ( B , fourth panel ), whereas that of mIL-1β was not ( B , second panel ). MDP exerted a negligible effect on the production of pro-IL-1β ( B , first panel ) and pro-IL-18 ( B , third panel ). GAPDH was used as an internal control ( B , fifth panel ). Blots are representative of three independent experiments.

Article Snippet: The primary antibodies used for immunoblotting were as follows: rabbit monoclonal antibodies against phospho-p38 MAPK (D3F9, Cat. No. #4511), p38 MAPK (D13E1, Cat. No. #8690), and phospho-NF-κB p65 (93H1, Cat. No. #3033) (Cell Signaling Technology, Inc., Danvers, MA); rabbit polyclonal antibodies against NF-κB p65 (Cat. No. #3034, Cell Signaling Technology); biotinylated antibodies against IL-1β (Cat. No. BAF681) and IL-18 (Cat. No. BAF588) (R&D Systems, Inc., Minneapolis, MN); and mouse monoclonal antibodies against GAPDH (MAb 6C5) (Cat. No. 5G4, HyTest Ltd., Turku, Finland).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: Nature communications

Article Title: SHANK3 depletion leads to ERK signalling overdose and cell death in KRAS-mutant cancers.

doi: 10.1038/s41467-024-52326-1

Figure Lengend Snippet: Fig. 1 | SHANK3 depletion inhibits cell proliferation in vitro and in vivo in different cancer types driven by distinct KRAS mutations. a A cell proliferation screen following control (siCTRL, grey) or SHANK3 silencing (siSHANK3_2 (red) or siSHANK3_7 (blue)) in wild-type (WT) or KRAS-mutant pancreatic (PDAC), lung (NSCLC) and colorectal (CRC) cancer cell lines. ARPE-19, non-transformed retinal epithelial cells. Shown are the individual data points relative to control [the mean of the control is set to 1.0 by definition; data are mean ± s.d.; n = 3 (Panc10.05 siCTRL and H226 siSHANK3_2) or 4 (other samples) individually silenced wells; two-way ANOVA with Dunnett’s multiple comparisons test]. b Spheroid growth of siCTRL or siSHANK3 PANC-1 or A549 cells. Shown are representative images and quantifica- tion of spheroid area (mean ± s.d.; n = 3 independent experiments; statistical

Article Snippet: The following primary antibodies were used: SHANK3 (Cat. No. HPA003446, Atlas antibodies and Cat. no. sc-30193, Santa Cruz), GFP (Cat. no. ab1218, Abcam), KRAS (Cat. no. WH0003845M1, SigmaAldrich), GAPDH (Cat. no. 5G4-6C5, Hytest), HSP70 (Hsc70/Hsp73; Cat. no. ADI-SPA-815, Enzo), phopho-ERK1/2 (Thr202/Tyr204;Cat. no. 4370S, Cell Signalling), ERK1/2 (Cat. no. 91025, Cell Signalling), phospho-AKT (Ser473) (Cat. no. 9271, Cell Signalling), AKT (Cat. no. 9272, Cell Signalling) and cleaved-PARP1 (Cat. no. ab4830andab32064 [E51], both from Abcam; recognise a larger 89 KD and a smaller 28 KD band, respectively).

Techniques: In Vitro, In Vivo, Control, Mutagenesis, Transformation Assay

Journal: Frontiers in Immunology

Article Title: Establishment and characterization of an immortalized red river hog blood-derived macrophage cell line

doi: 10.3389/fimmu.2024.1465952

Figure Lengend Snippet: Phosphorylation of p65 NF-κB and p38 MAPK, and the production of IL-1β and IL-18 in RZJ/IBM cells in response to LPS or MDP. The treatment with LPS or MDP induced the phosphorylation of NF-κB p65 and p38 MAPK in a dose-dependent manner ( A , first and third panels ). The equivalent protein loading of these molecules was confirmed by immunoblotting with anti-NF-κB p65 or anti-p38 MAPK antibodies ( A , second and fourth panels ). The dose-dependent production of pro-IL-1β and pro-IL-18 was also detected in cell lysates ( B , first and third panels ) or culture supernatants ( B , second and fourth panels ) from RZJ/IBM cells that had been stimulated with LPS for 3 days. The secretion of mIL-18 from LPS-treated RZJ/IBM cells into the culture supernatant was also detected ( B , fourth panel ), whereas that of mIL-1β was not ( B , second panel ). MDP exerted a negligible effect on the production of pro-IL-1β ( B , first panel ) and pro-IL-18 ( B , third panel ). GAPDH was used as an internal control ( B , fifth panel ). Blots are representative of three independent experiments.

Article Snippet: The primary antibodies used for immunoblotting were as follows: rabbit monoclonal antibodies against phospho-p38 MAPK (D3F9, Cat. No. #4511), p38 MAPK (D13E1, Cat. No. #8690), and phospho-NF-κB p65 (93H1, Cat. No. #3033) (Cell Signaling Technology, Inc., Danvers, MA); rabbit polyclonal antibodies against NF-κB p65 (Cat. No. #3034, Cell Signaling Technology); biotinylated antibodies against IL-1β (Cat. No. BAF681) and IL-18 (Cat. No. BAF588) (R&D Systems, Inc., Minneapolis, MN); and mouse monoclonal antibodies against GAPDH (MAb 6C5) (Cat. No. 5G4, HyTest Ltd., Turku, Finland).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: bioRxiv

Article Title: Targeting a broad spectrum of KRAS -mutant cancers by hyperactivation-induced cell death

doi: 10.1101/2022.09.21.508660

Figure Lengend Snippet: Left: SHANK 3 gene expression (mRNA levels) showing the efficiency of SHANK3 silencing in control or doxycycline-induced (Dox: +; 72 h) shSHANK3 expressing PANC-1 clones (clones 1C and 4S). Right: Representative immunoblots of the indicated proteins in control or doxycycline-induced sh SHANK3 expressing PANC-1 clones collected three days after induction. Samples were resolved and blotted on duplicate membranes (m#1 and m#2). p-ERK, phospho-ERK1/2 (Thr202/Y204); ERK, total ERK; AKT, total AKT; p-AKT, phospho-AKT S473; cleaved-PARP1, indicative of apoptosis; GAPDH, a loading control.

Article Snippet: The following primary antibodies were used: SHANK3 (Cat. No. HPA003446, Atlas antibodies and Cat. no. sc-30193, Santa Cruz), GFP (Cat. no. ab1218, Abcam), KRAS (Cat. no. WH0003845M1, Sigma-Aldrich), GAPDH (Cat. no. 5G4-6C5, Hytest), HSP70 (Hsc70/Hsp73; Cat. no. ADI-SPA-815, Enzo), phopho-ERK1/2 (Thr202/Tyr204) (Cat. no. 4370S, Cell Signaling), ERK1/2 (Cat. no. 91025, Cell Signaling), phospho-AKT (Ser473) (Cat. no. 9271, Cell Signaling), AKT (Cat. no. 9272, Cell Signaling) and cleaved-PARP1 (Cat. no. ab4830, Abcam).

Techniques: Gene Expression, Control, Expressing, Clone Assay, Western Blot