xaf1  (Boster Bio)

Journal: Journal of translational medicine

Article Title: Potential of CLSPN as a therapeutic target in melanoma: a key player in melanoma progression and tumor microenvironment.

doi: 10.1186/s12967-025-06455-w

Figure Lengend Snippet: Fig. 4 RNA sequencing of A2058 cells to investigate the function of CLSPN. (A) Volcano plot of the differential gene expression between CLSPN knock down group and the control group. The red points represented significantly up-regulated gene, and the blue points represented significantly down-reg ulated gene. (B) The top 20 enrichment of KEGG pathway terms analysis for the differential genes.(C) Protein-protein interaction analysis of differentially expressed functional genes. (D) Molecular docking result of CLSPN/IFI44L. (E) The mRNA expression of IFI44L and downstream genes after CLSPN knock down in A2058 by qPCR. (F) The protein levels of CLSPN, IFI44L, p-STAT1, STAT1 and XAF1 after si-CLSPN transfection by western blot. (G) The quantifica tion results of western blot by Image J (n = 3). Data were expressed as mean ± SD. * p < 0.05 and ** p < 0.01

Article Snippet: The membranes were blocked using 5% non-fat milk for 1 h, incubated overnight with primary antibodies against CLSPN (Affinity, DF7525), IFI44L (SAB, 27948), p-STAT1 (Proteintech, 28977-1-AP), STAT1 (Proteintech, 10144-2-AP), XAF1 (BOSTER, BA4986), GAPDH (Affinity, AF7021), antiBax (Cell Signaling Technology, #5023), anti-Bcl-2 (Cell Signaling Technology, #3498), anti-cleaved-PARP (Cell Signaling Technology, #5625T), anti-cleaved-caspase 9 (Cell Signaling Technology, #20750), anti-β-tubulin (Affinity, AF7011), anti-β-actin (Affinity, #AF7018) at 4 °C, and then incubated for 1 h with secondary antibody goat Anti-Rabbit IgG (Affinity, #S0001).

Techniques: RNA Sequencing, Gene Expression, Knockdown, Control, Functional Assay, Expressing, Transfection, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: SARM1 deficiency up-regulates XAF1, promotes neuronal apoptosis, and accelerates prion disease

doi: 10.1084/jem.20171885

Figure Lengend Snippet: SARM1 deficiency and prion infection up-regulated XAF1 expression in mouse brains. (A and B) Volcano plots of RNaseq data for uninfected (A) and RML6-infected (112 dpi; B) SARM1 −/− and WT mouse brains. Significants (red dots) are genes that show log2 ratios >1 or < −1 (fold change >2) with P < 0.01. (C) qRT-PCR of Xaf1 in uninfected and RML6-infected (112 dpi) SARM1 −/− , SARM1 +/– , and SARM1 +/+ mouse brains. n = 3–4 for each group. Relative expression was normalized to GAPDH expression and represented as the percentage of average values in uninfected SARM1 +/+ mice. (D and E) qRT-PCR of Sarm1 (D) and SARM1 Western blot (E) in SARM1 siRNA-treated CAD5 cells. (F) qRT-PCR of Xaf1 in SARM1 siRNA-treated CAD5 cells. n = 6 for each treatment. Relative expression was normalized to GAPDH expression and represented as the percentage of average values in nontarget control. (G) Upper panel: Images of in situ hybridization using 3-Plex negative probes. Middle and lower panels: Representative images of Sarm1, Xaf1, and Syp in situ hybridization in RML6-infected WT (middle) and SARM1 −/− (lower) mouse cerebella, hippocampi, and neocortex at 112 dpi. Differential interference contrast (DIC) and DAPI staining show the morphology and nuclei of cells. gl, granular layer; ml, molecular layer; pl, Purkinje cell layer. White arrows indicate Sarm1, Xaf1, and Syp triple-positive cells. Bars, 20 µm. qRT-PCR and Western blot results represent at least three independent experiments. In situ hybridization represents two independent experiments. n.s., P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Data are shown as mean ± SEM.

Article Snippet: To detect XAF1 in mouse brains and CAD5 cells, primary antibodies rabbit anti-mouse XAF1 antibody (1:1,000; ab17204 and ab81353 [Abcam]; OAAB14914 [Aviva Systems Biology]; A03432 [Boster]; PA1-41099 [Invitrogen]; GTX51339 [GeneTex]; NB100-56355 [Novus Biologicals]) were used.

Techniques: Infection, Expressing, Quantitative RT-PCR, Western Blot, Control, In Situ Hybridization, Staining

Journal: The Journal of Experimental Medicine

Article Title: SARM1 deficiency up-regulates XAF1, promotes neuronal apoptosis, and accelerates prion disease

doi: 10.1084/jem.20171885

Figure Lengend Snippet: SARM1 deficiency enhanced apoptosis in prion-infected SARM1 −/− brains. (A and B) Caspase 3 (A) and caspase 9 (B) activity assay of RML6-infected SARM1 −/− , SARM1 +/− , and SARM1 +/+ mouse brains at 112 dpi. n = 8 for SARM1 −/− and n = 4 for SARM1 +/− and SARM1 +/+ mice. Relative caspase 3 and caspase 9 activity was represented as the percentage of average values in SARM1 +/+ mice. (C) Left: TUNEL staining of brain tissues from RML6-infected SARM1 −/− , SARM1 +/− , and SARM1 +/+ mice at 112 dpi. Right: Quantification of TUNEL-positive cells. n = 7 for SARM1 −/− and n = 3 for SARM1 +/− and SARM1 +/+ mice. Bars, 10 µm. (D) Upper: Western blot for synapsin I on brains collected from terminally sick RML6-infected C57BL/6 mice and NBH-inoculated C57BL/6 mice. Lower: Densitometric quantification of the synapsin I Western blot showed a significant reduction of synapsin I in RML6-infected brains. n = 5 for NBH-treated and RML6-treated mice. (E) Left: Western blot for synapsin I in uninfected SARM1 −/− , SARM1 +/− , and SARM1 +/+ mice. Right: Densitometric quantification of the synapsin I Western blots showed unchanged synapsin I level in uninfected SARM1 −/− , SARM1 +/− , and SARM1 +/+ mice. n = 3 for SARM1 +/− and SARM1 +/+ mice; n = 5 for SARM1 −/− mice. (F) Left: Western blot for synapsin I in RML6-infected SARM1 −/− , SARM1 +/− , and SARM1 +/+ mouse brains at 112 dpi. Right: Densitometric quantification of the synapsin I Western blot. n = 3 for each genotype. (G) Caspase 3 activity assay of WT CAD5 and XAF1 KO CAD5 cell lysates after SARM1 siRNAs and TNFα treatments. (H) Caspase 9 activity assay of WT CAD5 and XAF1 KO CAD5 cell lysates after SARM1 siRNAs and TNFα treatments. n = 3. Relative caspase 3 and caspase 9 activity was represented as the percentage of average values in PBS groups. (I) RealTime-Glo assay of live WT CAD5 and XAF1 KO CAD5 cells after SARM1 siRNAs and TNFα treatments. n = 6. Relative cell numbers were represented as the percentage of average values in PBS groups. Relative caspase 3 and caspase 9 activity measurement represents two independent experiments. (J) Sequencing results of the Xaf1 gene in XAF1 KO CAD5 cells showed that the coding sequence of all sequenced clones was frameshifted. PAM, protospacer adjacent motif. Western blot results represent at least three independent experiments. Relative signal intensity of Western blot was represented as the percentage of average values in NBH-treated or SARM1 +/+ mice. n.s., P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Data are shown as mean ± SEM.

Article Snippet: To detect XAF1 in mouse brains and CAD5 cells, primary antibodies rabbit anti-mouse XAF1 antibody (1:1,000; ab17204 and ab81353 [Abcam]; OAAB14914 [Aviva Systems Biology]; A03432 [Boster]; PA1-41099 [Invitrogen]; GTX51339 [GeneTex]; NB100-56355 [Novus Biologicals]) were used.

Techniques: Infection, Activity Assay, TUNEL Assay, Staining, Western Blot, Caspase-3 Activity Assay, Glo Assay, Sequencing, Clone Assay

Journal: bioRxiv

Article Title: SARM1 deficiency, which prevents Wallerian degeneration, upregulates XAF1 and accelerates prion disease

doi: 10.1101/485052

Figure Lengend Snippet: (A) Volcano plots of RNAseq data for uninfected (A) and RML6-infected (112dpi) (B) SARM1 −/− and WT mouse brains. Significants (red dots) are genes that show log2 ratio >1 or <−1 (fold change>2) with p<0.01. (C) qRT-PCR of Xaf1 in uninfected and RML6-infected (112dpi) SARM1 −/− , SARM1 +/− and SARM1 +/+ (WT) mouse brains. n= 3-4 for each group. Relative expression was normalized to GAPDH expression and represented as percentage of average values in uninfected SARM1 +/+ mice. (D-E) qRT-PCR of Sarm1 (D) and SARM1 Western blot (E) in SARM1 siRNA treated CAD5 cells. (F) qRT-PCR of Xaf1 in SARM1 siRNA treated CAD5 cells. n=6 for each treatment. Relative expression was normalized to GAPDH expression and represented as percentage of average values in non-target control. (G) Upper panel: images of in situ hybridization using 3-Plex negative probes. Middle and lower panels: representative images of Sarm1, Xaf1 and Syp in situ hybridization in RML6-infected WT (middle) and SARM1 −/− (lower) mouse cerebella, hippocampi and neocortex at 112dpi. Differential interference contrast (DIC) and DAPI staining show the morphology and nuclei of cells. gl: granular layer; ml: molecular layer, pl: Purkinje cell layer. Lower panel: White arrows indicate Sarm1, Xaf1 and synaptophysin triple positive cells. Scale bars: 20μm. qRT-PCR and Western blot results represent at least three independent experiments. In situ hybridization represents two independent experiments.

Article Snippet: To detect XAF1 in mouse brains and CAD5 cells, primary antibodies rabbit anti–mouse XAF1 antibody (1:1000; Abcam, ab17204 and ab81353; Aviva Systems Biology, OAAB14914; Boster, A03432; Invitrogen, PA1-41099; GeneTex, GTX51339; Novus Biologicals, NB100-56355) were used.

Techniques: Infection, Quantitative RT-PCR, Expressing, Western Blot, Control, In Situ Hybridization, Staining

Journal: bioRxiv

Article Title: SARM1 deficiency, which prevents Wallerian degeneration, upregulates XAF1 and accelerates prion disease

doi: 10.1101/485052

Figure Lengend Snippet: (A-B) Caspase 3 (A) and caspase 9 (B) activity assay of RML6-infected SARM1 −/− , SARM1 +/− and SARM1 +/+ (WT) mouse brains at 112 dpi. n= 8 for SARM1 −/− and n=4 for SARM1 +/− and SARM1 +/+ (WT) mice. Relative caspase 3 and caspase 9 activity was represented as percentage of average values in SARM1 +/+ mice. (C) Left: TUNEL staining of brain tissues from RML6-infected SARM1 −/− , SARM1 +/− and SARM1 +/+ (WT) mice at 112 dpi. Right: Quantification of TUNEL positive cells. n=7 for SARM1 −/− and n=3 for SARM1 +/− and SARM1 +/+ (WT) mice. Scale bars: 10μm. (D) Left: Western blot for synapsin I on brains collected from terminally sick RML6-infected C57BL/6 mice and age-matched NBH-inoculated C57BL/6 mice. Right: Densitometric quantification of the synapsin I Western blot showed significant reduction of synapsin I in RML6-infected brains. n=5 for NBH-treated and RML6-treated mice. Relative signal intensity was represented as percentage of average values in NBH-treated mice. (E) Left: Western blot for synapsin I in uninfected SARM1 −/− , SARM1 +/− and SARM1 +/+ (WT) mice. Right: Densitometric quantification of the synapsin I Western blots showed unchanged synapsin I level in uninfected SARM1 −/− , SARM1 +/− and SARM1 +/+ (WT) mice. n=3 for SARM1 +/− and SARM1 +/+ (WT) mice, n=5 for SARM1 −/− mice. Relative signal intensity was represented as percentage of average values in SARM1 +/+ mice. (F) Left: Western blot for synapsin I in RML6-infected SARM1 −/− , SARM1 +/− and SARM1 +/+ (WT) mouse brains at 112 dpi. Right: Densitometric quantification of the synapsin I Western blot. n=3 for each genotype. The relative signal intensity was represented as percentage of average values in SARM1 +/+ mice. (G) Caspase 3 activity assay of wild type (WT) CAD5 and XAF1 KO CAD5 cell lysates after SARM1 siRNAs and TNFα treatments. (H) Caspase 9 activity assay of WT CAD5 and XAF1 KO CAD5 cell lysates after SARM1 siRNAs and TNFα treatments. n=3. Relative caspase 3 and caspase 9 activity was represented as percentage of average values in PBS groups. (I) RealTime-Glo assay of live WT CAD5 and XAF1 KO CAD5 cells after SARM1 siRNAs and TNFα treatments. n=6. Relative cell numbers were represented as percentage of average values in PBS groups. Relative caspase 3 and caspase 9 activity measurement represents two independent experiments. (J) Sequencing results of Xaf1 gene in XAF1 KO CAD5 cells showed that the coding sequence of all sequenced clones was frameshifted. Western blot results represent at least three independent experiments.

Article Snippet: To detect XAF1 in mouse brains and CAD5 cells, primary antibodies rabbit anti–mouse XAF1 antibody (1:1000; Abcam, ab17204 and ab81353; Aviva Systems Biology, OAAB14914; Boster, A03432; Invitrogen, PA1-41099; GeneTex, GTX51339; Novus Biologicals, NB100-56355) were used.

Techniques: Activity Assay, Infection, TUNEL Assay, Staining, Western Blot, Caspase-3 Activity Assay, Glo Assay, Sequencing, Clone Assay

Journal: bioRxiv

Article Title: SARM1 deficiency, which prevents Wallerian degeneration, upregulates XAF1 and accelerates prion disease

doi: 10.1101/485052

Figure Lengend Snippet: (A) Volcano plots of RNAseq data for uninfected (A) and RML6-infected (112dpi) (B) SARM1 −/− and WT mouse brains. Significants (red dots) are genes that show log2 ratio >1 or <−1 (fold change>2) with p<0.01. (C) qRT-PCR of Xaf1 in uninfected and RML6-infected (112dpi) SARM1 −/− , SARM1 +/− and SARM1 +/+ (WT) mouse brains. n= 3-4 for each group. Relative expression was normalized to GAPDH expression and represented as percentage of average values in uninfected SARM1 +/+ mice. (D-E) qRT-PCR of Sarm1 (D) and SARM1 Western blot (E) in SARM1 siRNA treated CAD5 cells. (F) qRT-PCR of Xaf1 in SARM1 siRNA treated CAD5 cells. n=6 for each treatment. Relative expression was normalized to GAPDH expression and represented as percentage of average values in non-target control. (G) Upper panel: images of in situ hybridization using 3-Plex negative probes. Middle and lower panels: representative images of Sarm1, Xaf1 and Syp in situ hybridization in RML6-infected WT (middle) and SARM1 −/− (lower) mouse cerebella, hippocampi and neocortex at 112dpi. Differential interference contrast (DIC) and DAPI staining show the morphology and nuclei of cells. gl: granular layer; ml: molecular layer, pl: Purkinje cell layer. Lower panel: White arrows indicate Sarm1, Xaf1 and synaptophysin triple positive cells. Scale bars: 20μm. qRT-PCR and Western blot results represent at least three independent experiments. In situ hybridization represents two independent experiments.

Article Snippet: Supplementary figure 2 (A-G) Western blots for XAF1 in CAD5 cell lysates using various anti-XAF1 antibodies: (A) Abcam (ab17204); (B) Abcam (ab81353); (C) Aviva System Biology (OAAB14914); (D) Boster (A03432); (E) GeneTex (GTX51339); (F) Invitrogene (PA1-41099); (G) Novus Biologicals (NB100-56355).

Techniques: Infection, Quantitative RT-PCR, Expressing, Western Blot, Control, In Situ Hybridization, Staining

Journal: bioRxiv

Article Title: SARM1 deficiency, which prevents Wallerian degeneration, upregulates XAF1 and accelerates prion disease

doi: 10.1101/485052

Figure Lengend Snippet: (A-B) Caspase 3 (A) and caspase 9 (B) activity assay of RML6-infected SARM1 −/− , SARM1 +/− and SARM1 +/+ (WT) mouse brains at 112 dpi. n= 8 for SARM1 −/− and n=4 for SARM1 +/− and SARM1 +/+ (WT) mice. Relative caspase 3 and caspase 9 activity was represented as percentage of average values in SARM1 +/+ mice. (C) Left: TUNEL staining of brain tissues from RML6-infected SARM1 −/− , SARM1 +/− and SARM1 +/+ (WT) mice at 112 dpi. Right: Quantification of TUNEL positive cells. n=7 for SARM1 −/− and n=3 for SARM1 +/− and SARM1 +/+ (WT) mice. Scale bars: 10μm. (D) Left: Western blot for synapsin I on brains collected from terminally sick RML6-infected C57BL/6 mice and age-matched NBH-inoculated C57BL/6 mice. Right: Densitometric quantification of the synapsin I Western blot showed significant reduction of synapsin I in RML6-infected brains. n=5 for NBH-treated and RML6-treated mice. Relative signal intensity was represented as percentage of average values in NBH-treated mice. (E) Left: Western blot for synapsin I in uninfected SARM1 −/− , SARM1 +/− and SARM1 +/+ (WT) mice. Right: Densitometric quantification of the synapsin I Western blots showed unchanged synapsin I level in uninfected SARM1 −/− , SARM1 +/− and SARM1 +/+ (WT) mice. n=3 for SARM1 +/− and SARM1 +/+ (WT) mice, n=5 for SARM1 −/− mice. Relative signal intensity was represented as percentage of average values in SARM1 +/+ mice. (F) Left: Western blot for synapsin I in RML6-infected SARM1 −/− , SARM1 +/− and SARM1 +/+ (WT) mouse brains at 112 dpi. Right: Densitometric quantification of the synapsin I Western blot. n=3 for each genotype. The relative signal intensity was represented as percentage of average values in SARM1 +/+ mice. (G) Caspase 3 activity assay of wild type (WT) CAD5 and XAF1 KO CAD5 cell lysates after SARM1 siRNAs and TNFα treatments. (H) Caspase 9 activity assay of WT CAD5 and XAF1 KO CAD5 cell lysates after SARM1 siRNAs and TNFα treatments. n=3. Relative caspase 3 and caspase 9 activity was represented as percentage of average values in PBS groups. (I) RealTime-Glo assay of live WT CAD5 and XAF1 KO CAD5 cells after SARM1 siRNAs and TNFα treatments. n=6. Relative cell numbers were represented as percentage of average values in PBS groups. Relative caspase 3 and caspase 9 activity measurement represents two independent experiments. (J) Sequencing results of Xaf1 gene in XAF1 KO CAD5 cells showed that the coding sequence of all sequenced clones was frameshifted. Western blot results represent at least three independent experiments.

Article Snippet: Supplementary figure 2 (A-G) Western blots for XAF1 in CAD5 cell lysates using various anti-XAF1 antibodies: (A) Abcam (ab17204); (B) Abcam (ab81353); (C) Aviva System Biology (OAAB14914); (D) Boster (A03432); (E) GeneTex (GTX51339); (F) Invitrogene (PA1-41099); (G) Novus Biologicals (NB100-56355).

Techniques: Activity Assay, Infection, TUNEL Assay, Staining, Western Blot, Caspase-3 Activity Assay, Glo Assay, Sequencing, Clone Assay