Journal: PloS one

Article Title: Identification of PBX1 target genes in cancer cells by global mapping of PBX1 binding sites.

doi: 10.1371/journal.pone.0036054

Figure Lengend Snippet: Figure 6. MEOX1 interacts with PBX1 and mediates its growth effect. A. HEK293 cells were transfected with PBX1-V5 and/or MEOX1-FLAG expression vectors. Immunoprecipitation and Western blot were performed using epitope tag-specific antibodies. B. Left panel: OVCAR3 cells were transfected with MEOX1 expression vector or control vector. Western blot was performed to test MEOX1 expression (top). The same cells were transfected with PBX1 siRNA or control siRNA and Western blot was performed to test the down-regulation of PBX1 protein (middle). Detection of GAPDH protein was used as a loading control (bottom). Right panel: Relative cell numbers were measured in OVCAR3 cells transfected with MEOX1 cDNA, PBX1 siRNA, control plasmid (pLPC), and control siRNA (siLuc). Student’s t-test was used to determine the significance between the MEOX1 over-expressed group and the control group. doi:10.1371/journal.pone.0036054.g006

Article Snippet: Western blot was performed using an anti-FLAG antibody (Sigma, St. Louis, MO) to detect MEOX1 protein or an anti-V5 antibody (Bethyl, Montgomery, TX) to detect PBX1 protein.

Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot, Plasmid Preparation, Control

Journal: European journal of immunology

Article Title: A common human TLR1 polymorphism regulates the innate immune response to lipopeptides.

doi: 10.1002/eji.200737034

Figure Lengend Snippet: Figure 2. Immunofluorescence and expression studies of TLR1 variants. (A) TLR1 coding region and SNP positions; LRR, leucine-rich region; TM, transmembrane domain. SNP N248S and I602S are marked with a circle. (B–F) Localization patterns of V5-epitope-tagged TLR1 variants that were transfected into HEK293 cells and visualized by immunofluorescence. The TLR1 constructs varied at amino acids 248 (N or S) and 602 (I or S) and include TLR1_NI (248S_602I) (B), NS (248N_602S) (C), SS (248S_602S) (D), SI (248S_602I) (E), and a pEF6 empty vector control (F). (G) HEK293 cells were transfected with control vector (pEF6 without insert; lane 1), TLR1_NI (lane 2), TLR1_NS (lane 3), or TLR1_SS (lane 4), or TLR1_SI (lane 5); Western immunoblot probed with an anti-V5 antibody.

Article Snippet: The cells were subsequently incubated with an anti-V5 epitope antibody (Serotec, Oxford, UK) followed by a FITC-conjugated rabbit anti-mouse IgG (Serotec), and thenmounted in mounting medium, containing 25% w/v glycerol, 10% w/v polyvinyl alcohol (MW 30 000–70 000; Sigma-Aldrich), 0.1 M Tris-Cl pH 8.5, and 2.5% 1,4-diazabicyclo-[2.2.2]-octane (DABCO).

Techniques: Immunofluorescence, Expressing, Transfection, Construct, Plasmid Preparation, Control, Western Blot

Journal: Nature medicine

Article Title: RAF inhibitor PLX8394 selectively disrupts BRAF-dimers and RAS-independent BRAF mutant-driven signaling

doi: 10.1038/s41591-018-0274-5

Figure Lengend Snippet: (a) Immunoprecipitation (IP) assays were performed with anti-V5 agarose on cell lysates from 293H (NRAS Q61K) cells that ectopically co-express BRAF-V5 and BRAF-FLAG, p61 BRAF-V5 and p61 BRAF-FLAG, CRAF-V5 and BRAF-FLAG, catC-V5 and p61 BRAF-FLAG, CRAF-V5 and CRAF-FLAG or catC-V5 and catC-FLAG. Prior to the IP experiment, cells were treated with increasing doses of PLX8394 for 1hr as indicated. The immunoprecipitated proteins were assayed by Western blot with anti-V5 and anti-FLAG antibody (left panel). The interaction between FLAG and V5 tagged proteins were represented by the levels of FLAG tagged and V5 tagged proteins pulled down, quantified by densitometry and normalized to levels in the untreated cells. The curves of protein interactions in response to the drug treatments were generated using Prism6 (right panel). The dots represent the relative FLAG tagged protein and V5 tagged protein interaction levels as indicated. The experiments were repeated 3 times, independently. (b) The indicated pairs of FLAG- and V5-tagged proteins were ectopically co-expressed in 293H cells for 24hrs. Then the cells were treated with 1μM PLX8394 for 1hr. IPs were then performed on lysates from these cells with anti-FLAG agarose. The Input and IP samples were assayed by Western blot with anti-FLAG/V5 antibodies as indicated. The experiments were repeated 4 times, independently. (c) Dimer interfaces highlighting the key structural differences between BRAF and CRAF. If PLX8394 binds to the promoter on the left (Protomer 1), its interaction with L505 (BRAF) or L397 (CRAF) is expected to push the C-terminal end of αC helix out, thereby perturbing the interactions formed by the two basic residues (R506 and K507 in BRAF and R398 and K399 in CRAF). Whether this structural alteration results in dimer disruption is determined by the partner protomer (Protomer 2). If Protomer 2 is CRAF, Y340 forms a strong cation-π interaction with R506 (BRAF) or R398 (CRAF). The interface is further stabilized by the interaction between the backbone of Y341 and K507 (BRAF) or K399 (CRAF). The energy barrier to overcome these concerted set of interactions is high. However, if Protomer 2 is BRAF, D448 forms a salt bridge with R506 (BRAF) or R398 (CRAF). The backbone of the D449 is in a conformation not conducive to interaction with K507 (BRAF) or K399 (CRAF), and the energy barrier to break the dimer is lower. (d) and (e) The indicated FLAG- and V5-tagged proteins were ectopically expressed in 293H cells for 24hrs. Then the cells were treated with 1μM PLX8394 for 1hr. The cells were lysed and the lystates were subjected to IP with anti-V5 agarose. The Input and IP samples were assayed by Western blot with anti-FLAG/V5 antibodies. These experiments were all repeated 3 times, independently. For gel source data, see .

Article Snippet: Antibodies used include: anti-p217/p221-MEK1/2 (p-MEK1/2) (#9154, lot#14 ), anti-p202/p204-ERK1/2 (p-ERK1/2) (#4370, lot# 17), anti-MEK1/2 (#4694, lot# 6), and anti-ERK1/2 (#4696, lot# 16) from Cell Signaling; anti-V5 (46–1157, lot# 1902786) from Invitrogen; anti-BRAF (cat# sc-5284, lot# C0116), anti-Cyclin D1 (cat# sc-718, lot# G1411 ) and anti-ARAF (cat# sc-166771, lot# A2617 ) from Santa Cruz Biotechnology; anti-FLAG (cat# F1804, lot# SLBT7654) from Sigma; anti-CRAF (cat# 610152, lot#7208706) from BD Transduction Laboratories; and anti-RAS (cat# 1862335, Lot # S1259698) from the active RAS pull-down and detection kit (Thermo Fisher Scientific, cat# 16117).

Techniques: Immunoprecipitation, Western Blot, Generated