ip lysis buffer  (Thermo Fisher)

Journal: International Journal of Surgery (London, England)

Article Title: LINC00476 cooperates with ARIH2 and suppresses pancreatic cancer progression by inducing VIM ubiquitination

doi: 10.1097/JS9.0000000000003596

Figure Lengend Snippet: LINC00476 binds to VIM. (A) KEGG pathway analysis of LINC00476 based on transcriptome sequencing. (B) FISH staining showed that LINC00476 (red) was mainly localized in the cytoplasm and cytoskeleton. DAPI (blue) was used for nuclear counterstaining. (C) RNA pull-down assay was performed to detect the binding protein with biotinylated LINC00476. LINC00476 antisense RNA was used as the negative control. (D) GO-CC (cellular component) analysis predicting the potential proteins binding to LINC00476. (E) Western blotting was performed to evaluate the specific association of VIM with biotinylated LINC00476. Lysates of AsPC-1 and PANC-1 cells were harvested for RNA pull-down assays. LINC00476 antisense RNA was used as the negative control. (F and G) RNA-binding protein immunoprecipitation (RIP) assay was performed using an antibody against VIM. QRT-PCR was used to detect LINC00476 enrichment. IgG was used as the isotype control. Two-tailed Student’s t -test. (H) FISH staining showed that LINC00476 (red) was colocalized with VIM (green). (I) Secondary structure of LINC00476 ( http://www.Lncipedia.Org/ ). (J) Western blotting of VIM in samples precipitated by biotinylated full-length LINC00476 (FL) or LINC00476 truncations (Δ1: 1–485 bp; Δ2: 486–1024 bp; Δ3: 1025–1270bp). FL LINC00476 was used as the positive control. (K) Flag-RIP assay for LINC00476 showing its fold enrichment in cells transiently transfected with plasmids containing Flag-tagged full-length and truncated VIM constructs (Del1: 1–101 aa; Del2: 101–410 aa; Del3: 101–466 aa). IgG-RIP was used as the internal control. P values are shown as *** P < 0.001; **** P < 0.0001. All data represent the means ± SD from three independent assays.

Article Snippet: Initially, cells were transfected with a specific plasmid, and the transfected cells were subsequently lysed in an immunoprecipitation (IP) lysis buffer solution (HY-K1002, MCE, USA).

Techniques: Sequencing, Staining, Pull Down Assay, Binding Assay, Negative Control, Western Blot, RNA Binding Assay, Immunoprecipitation, Quantitative RT-PCR, Control, Two Tailed Test, Positive Control, Transfection, Construct

Journal: International Journal of Surgery (London, England)

Article Title: LINC00476 cooperates with ARIH2 and suppresses pancreatic cancer progression by inducing VIM ubiquitination

doi: 10.1097/JS9.0000000000003596

Figure Lengend Snippet: ARIH2 is the E3 ligases for VIM. (A) Venn diagram for identifying nine candidate E3 ligases overlapping with the potential binding proteins of LINC00476. (B) The protein level of ARIH2 in LINC00476 overexpression and knockout cells. (C and D) A RNA-binding protein immunoprecipitation (RIP) assay was performed using an antibody against VIM. QRT-PCR was used to detect LINC00476 and VIM enrichment. IgG was used as the isotype control. Two-tailed Student’s t -test. (E) Western blotting was performed to evaluate the specific association of ARIH2 with biotinylated LINC00476. Lysates of AsPC-1 and MIA PaCa-2 cells were harvested for RNA pull-down assays. LINC00476 antisense RNA was used as the negative control. ( F ) FISH staining showed that LINC00476 (red) was colocalized with ARIH2 (yellow) and VIM (green) in the LINC00476 overexpression and knockout cells. DAPI (blue) was used for nuclear counterstaining. P values are shown as **** P < 0.0001. All data represent the means ± SD from three independent assays.

Article Snippet: Initially, cells were transfected with a specific plasmid, and the transfected cells were subsequently lysed in an immunoprecipitation (IP) lysis buffer solution (HY-K1002, MCE, USA).

Techniques: Binding Assay, Over Expression, Knock-Out, RNA Binding Assay, Immunoprecipitation, Quantitative RT-PCR, Control, Two Tailed Test, Western Blot, Negative Control, Staining

Journal: International Journal of Surgery (London, England)

Article Title: LINC00476 cooperates with ARIH2 and suppresses pancreatic cancer progression by inducing VIM ubiquitination

doi: 10.1097/JS9.0000000000003596

Figure Lengend Snippet: ARIH2 is necessary for LINC00476 induced VIM ubiquitination. (A) The protein levels of VIM and ARIH2 were determined by Western blotting. LINC00476 overexpression and KO3 cells treated with MG132 for 4 h before cell lysates were harvested for co-IP with an anti-VIM antibody and anti-ARIH2 antibody. (B and C) The ubiquitination levels of VIM were determined by Western blotting. AsPC-1 cells were transiently transfected with ARIH2 overexpression (B), MIA PaCa-2 cells transfected ARIH2 silencing (C) and also with the corresponding control plasmids and then treated with MG132 for 4 h before cell lysates were harvested for co-IP with an anti-VIM antibody and immunoblotted with an anti-HA antibody. (D) The protein levels of ARIH2 in MIA PaCa-2 and AsPC-1 cells treated with cycloheximide (CHX, 50 μg/mL) for 0, 1, 3, and 6 h. AsPC-1 cells transfected with ARIH2 overexpression (upper), MIA PaCa-2 cells transfected with ARIH2 silencing (lower). (E) The protein levels of ARIH2 in MIA PaCa-2 and AsPC-1 cells treated with MG132 (10 µM) for 0, 1, 3, and 6 h. AsPC-1 cells transfected with ARIH2 overexpression (upper), MIA PaCa-2 cells transfected with ARIH2 silencing (lower). (F) The ubiquitination levels of VIM in the products of IP with an anti-Flag antibody in lysates from LINC00476 knockout or ARIH2 overexpression and control plasmids. (G) Immunoprecipitation assay showing K29-mediated ubiquitination of VIM by ARIH2. The 293T cells were cotransfected with Flag-VIM, different linkages of HA-ubiquitin mutant (wild-type, K27R, K29R, K49R, and K63R), or His-ARIH2 plasmids; and then, cell lysates were immunoprecipitated with anti-Flag antibody. (H) Flag-VIM plasmid was cotransfected into 293T cells together with wild-type, K27, K29, or lysine mutant (K27R and K29R) of HA-Ubiquitin in the absence or presence of His-ARIH2 plasmid. Cell lysates were immunoprecipitated with anti-Flag antibody and then immunoblotted with the indicated antibodies. All data represent the means ± SD from three independent assays.

Article Snippet: Initially, cells were transfected with a specific plasmid, and the transfected cells were subsequently lysed in an immunoprecipitation (IP) lysis buffer solution (HY-K1002, MCE, USA).

Techniques: Ubiquitin Proteomics, Western Blot, Over Expression, Co-Immunoprecipitation Assay, Transfection, Control, Knock-Out, Immunoprecipitation, Mutagenesis, Plasmid Preparation