- PA5-25475 - Invitrogen Antibodies FBXO9 antibody

Qu X, Sun Z, Wang Y, Ong HS
PeerJ 2021;9:e12510

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis in mouse liver tissue lysates (15 µg per lane) using a FBXO9 polyclonal antibody (Product # PA5-25475).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of WiDr cells using a FBXO9 polyclonal antibody (Product # PA5-25475) (right) compared to a negative control cell (left) at a dilution of 1:10-50, followed by a FITC-conjugated goat anti-rabbit antibody

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: 10.7717/peerj.12510/fig-3 Figure 3 FBXO9 was downregulated in osteoclasts after ZA treatment. (A) Venn analysis of DEGs of alendronate and risedronate-treated osteoclast. (B and C) The mRNA level of 18 genes in Raw264.7 and BMDM derived osteoclasts was assessed using qPCR after treatment with ZA (50 muM) ( n = 3) * p < 0.05, ** p < 0.01. (D) The protein level of FBXO9 in Raw264.7 and BMDM derived osteoclasts was assessed using western blot after treatment with ZA (50 muM).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: 10.7717/peerj.12510/fig-4 Figure 4 FBXO9 inhibition facilitated the ferroptosis of osteoclasts. (A) The mRNA level of FBXO9 in BMDM derived osteoclasts was assessed using qPCR after treatment with or without si-FBXO9 ( n = 3). *** p < 0.001. (B) The protein level of FBXO9 in BMDM derived osteoclasts was assessed using western blot after treatment with or with out si-FBXO9 ( n = 3). ** p < 0.01. (C) Cell viability of BMDM derived osteoclasts was assessed using CCK8 assay after treatment with or without si-FBXO9 ( n = 3). ** p < 0.01. (D and E) Cell viability of BMDM derived osteoclasts was assessed using FDA staining after treatment with or without si-FBXO9 ( n = 3). * p < 0.05. (F-J) The level of Fe 2+ , MDA content, ROS level, the level of Gpx4, and GSH content in BMDM derived osteoclasts was assessed by Elisa assay after treatment with or without si-FBXO9 ( n = 3) * p < 0.05, ** p < 0.01.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: 10.7717/peerj.12510/fig-5 Figure 5 ZA treatment facilitated the ferroptosis of osteoclasts by suppressing FBXO9. (A) The mRNA level of FBXO9 in BMDM derived osteoclasts was assessed using qPCR after treatment with ZA (50 muM) in the presence or absence of FBXO9 ( n = 3). ** p < 0.01. (B) The protein level of FBXO9 in BMDM derived osteoclasts was assessed using western blot after treatment with ZA (50 muM) in the presence or absence of FBXO9 ( n = 3). * p < 0.05. (C) Cell viability of BMDM derived osteoclasts was assessed using CCK8 assay after treatment with ZA (50 muM) in the presence or absence of FBXO9 ( n = 3). * p < 0.05, ** p < 0.01. (D and E) Cell viability of BMDM derived osteoclasts was assessed using FDA staining after treatment with ZA (50 muM) in the presence or absence of FBXO9 ( n = 3). * p < 0.05. (F-J) The level of Fe 2+ , MDA content, ROS level, the level of Gpx4, and GSH content in BMDM derived osteoclasts was assessed by Elisa assay after treatment with ZA (50 muM) in the presence or absence of FBXO9 ( n = 3) * p < 0.05, ** p < 0.01, *** p < 0.001.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: 10.7717/peerj.12510/fig-6 Figure 6 FBXO9 inhibition facilitated the ferroptosis of osteoclasts by blocking the ubiquitin mediated-proteasome degradation of p53. (A) The target of FBXO9 was predicted by ubibrowser. (B) The p53 mRNA expression in the FBXO9 knockdown and control cell was assessed by qPCR ( n = 3). (C) The protein level of p53 in the FBXO9 knockdown and control cell was assessed by western blot ( n = 3). (D) FBXO9 directly interacts with p53. The proteins from BMDM derived osteoclasts were IP with IgG or antibodies against FBXO9 and p53, following by western blot analysis ( n = 3). (E) The stability of p53 protein was regulated by FBXO9. BMDM derived osteoclasts treated with or without si-FBXO9 in the presence of cycloheximide (CHX, 25 ug/ml) for various times as indicated and cell lysates were then assessed by western blot ( n = 3). ** p < 0.01. (F) The cell lysates isolated from scramble and si-FBXO9 infected BMDM derived osteoclasts were immunoprecipitated with anti-p53 antibody, then analyzed by western blot using ubiquitin antibody ( n = 3).

PA5-25475