Journal:

Article Title: Rab22a Regulates the Recycling of Membrane Proteins Internalized Independently of Clathrin V⃞

doi: 10.1091/mbc.E04-04-0342

Figure Lengend Snippet: GFP-Rab22a is not localized on the vacuoles accumulated in Arf6-Q67L–expressing cells. HeLa cells were cotransfected with GFP-Rab22a and either Arf6-wt (top), Arf6-Q67L (middle), or Arf6-T27N (bottom). Cells were probed with a polyclonal antiserum directed against Arf6 followed by the Alexa-594-goat anti rabbit IgG. Arrowheads point to region of the PM where Arf6 does not colocalize with GFP-Rab22a. Bar, 10 μm.

Article Snippet: The affinity-purified rabbit polyclonal antibody against Rab4 used for immunoblotting was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing

Journal:

Article Title: Rab22a Regulates the Recycling of Membrane Proteins Internalized Independently of Clathrin V⃞

doi: 10.1091/mbc.E04-04-0342

Figure Lengend Snippet: siRNA-mediated depletion of Rab22a inhibits MHCI recycling to the PM. (A) Lysates from either mock- or siRNA-treated cells were subjected to electrophoresis and probed with antibodies directed against Rab22a, Rab4, Rab5, Rab11, Arf6, TfnR, and actin. (B) Mock- (top) or siRNA-treated cells (bottom) were allowed to internalize antibody to MHCI and 633 Tfn and processed for confocal microscopy as described in MATERIALS AND METHODS. Arrowheads and asterisks point to MHCI-containing tubules and MHCI-containing vesicles, respectively. Bars, 10 μm. (C) Mock- or siRNA-treated cells were scored for either MHCI-containing tubules (closed bars) or MHCI-containing vesicles (open bars) as described in MATERIALS AND METHODS. Data are averages of two independent experiments ± SEM. (D) Recycling to the PM of MHCI and Tfn was measured as described in MATERIALS AND METHODS in either mock- (closed bars) or siRNA-treated (open bars) cells. Data are averages of two independent experiments ± SEM. Student's t-test was used to test the statistical significance of the data. For the recycling, the p values were mock versus siRNA, p < 0.001 for both MHCI and transferrin recycling.

Article Snippet: The affinity-purified rabbit polyclonal antibody against Rab4 used for immunoblotting was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Electrophoresis, Confocal Microscopy

Journal: Research in Veterinary Science

Article Title: EIF4A2 interacts with the membrane protein of transmissible gastroenteritis coronavirus and plays a role in virus replication

doi: 10.1016/j.rvsc.2018.12.005

Figure Lengend Snippet: Primers for QRT-PCR.

Article Snippet: The beta tubulin rabbit polyclonal antibody (β-tubulin) and EIF4A2 rabbit polyclonal antibody were purchased from Proteintech (USA).

Techniques: Sequencing

Journal: Research in Veterinary Science

Article Title: EIF4A2 interacts with the membrane protein of transmissible gastroenteritis coronavirus and plays a role in virus replication

doi: 10.1016/j.rvsc.2018.12.005

Figure Lengend Snippet: GST-pull down and CO-IP were used to analyze the interaction between the M protein and candidate protein EIF4A2 by SDS-PAGE and Western blot. (A) The results of the GST pull-down by SDS-PAGE M: Prestained protein marker; 1: purified GST-M fusion protein + purified His-EIF4A2 fusion protein; 2: purified GST-tag protein + purified His-EIF4A2 fusion protein; 3: purified His-EIF4A2 fusion protein; 4: purified GST-M fusion protein; 5: purified GST-tag protein; (B) The results of the GST pull-down by Western blot M: Prestained protein marker; 1–2: His-EIF4A2 fusion protein. (C) pIECs infected with TGEV and control cells were harvested at 24 h, and the M protein was precipitated by an antibody in TGEV-infected group but not the mock group. TGEV+ represent the pIECs infected with TGEV and TGEV- represent uninfected TGEV in pIECs.

Article Snippet: The beta tubulin rabbit polyclonal antibody (β-tubulin) and EIF4A2 rabbit polyclonal antibody were purchased from Proteintech (USA).

Techniques: Co-Immunoprecipitation Assay, SDS Page, Western Blot, Marker, Purification, Infection, Control

Journal: Research in Veterinary Science

Article Title: EIF4A2 interacts with the membrane protein of transmissible gastroenteritis coronavirus and plays a role in virus replication

doi: 10.1016/j.rvsc.2018.12.005

Figure Lengend Snippet: Co-localization of EIF4A2 with the M protein. Determining the location of the M protein using FITC-conjugated goat anti-rat IgG (green) as the secondary antibody after pIECs were infected with TGEV. The red colour indicates the distribution domain of EIF4A2. The nucleus was stained with DAPI (blue). A ZEISS LSM 800 with Airyscan was used to examine the triple-stained location. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The beta tubulin rabbit polyclonal antibody (β-tubulin) and EIF4A2 rabbit polyclonal antibody were purchased from Proteintech (USA).

Techniques: Infection, Staining

Journal: Research in Veterinary Science

Article Title: EIF4A2 interacts with the membrane protein of transmissible gastroenteritis coronavirus and plays a role in virus replication

doi: 10.1016/j.rvsc.2018.12.005

Figure Lengend Snippet: EIF4A2 gene silencing. pIECs were cultured in six-well culture plates and transfected with siRNAs (20 nM). The cell lysates were harvested for Western blotting at 48 h. The average densitometric intensity of EIF4A2 in the immunoblot analysis, with β-tubulin used as a control (A and B). The relativelevel of EIF4A2 mRNA expression was used to further evaluate the interference effect of siRNA2 by RT-qPCR at 36 h (C). *P < 0.05.

Article Snippet: The beta tubulin rabbit polyclonal antibody (β-tubulin) and EIF4A2 rabbit polyclonal antibody were purchased from Proteintech (USA).

Techniques: Cell Culture, Transfection, Western Blot, Control, Expressing, Quantitative RT-PCR

Journal: Research in Veterinary Science

Article Title: EIF4A2 interacts with the membrane protein of transmissible gastroenteritis coronavirus and plays a role in virus replication

doi: 10.1016/j.rvsc.2018.12.005

Figure Lengend Snippet: Virus titration. EIF4A2-knockdown with siRNA2 cells and negative control knockdown cells were adsorbed with TGEV (MOI = 0.1) at 37 °C for 1 h. The culture supernatants of the cells and the virus-associated cells infected with TGEV were collected at different points. The viral titers are presented as the averages from three independent samples. **P < 0.01 and ***P < 0.001.

Article Snippet: The beta tubulin rabbit polyclonal antibody (β-tubulin) and EIF4A2 rabbit polyclonal antibody were purchased from Proteintech (USA).

Techniques: Virus, Titration, Knockdown, Negative Control, Infection

Journal: bioRxiv

Article Title: Intraflagellar transport protein 74 is essential for mouse spermatogenesis and male fertility by regulating axonemal microtubule assembly in mice

doi: 10.1101/457804

Figure Lengend Snippet: A.Examination of selective IFT protein expression levels in the control and conditional Ift74 knockout mice by Western blot. Compared to the controls, the expression levels of IFT27, IFT57, IFT81, IFT88 and IFT140, but not IFT20 and IFT25 are reduced in the conditional Ift74 knockout mice. a: representative Western blot result; b: quantitative analysis of selective IFT protein expression. B. Examination of testicular expression levels of ODF2, a component of sperm tail outer dense fibers, AKAP4, a major component of fibrous sheath, and SPAG16L, a component of axonemal central apparatus protein in the control and conditional Ift74 knockout mice. There was no difference in ODF2 and SPAG16L expression. However, the processed AKAP4 was significantly reduced in the conditional Ift74 knockout mice. β-ACTIN was used as controls. a: representative Western blot result; b: quantitative analysis of ODF2 and SPAG16L; c: quantitative analysis of AKAP4.

Article Snippet: Then the Nonspecific sites were blocked in a Tris-buffered saline solution containing 5% nonfat dry milk powder and 0.05% Tween 20 (TBST) for 1 hour at room temperature, and the membranes were incubated with indicated primary antibodies (IFT25: 1:2000, Cat No: 15732-1-AP, ProteinTech; IFT74: 1:2000, Cat No: AAS27620e from ANTIBODY VERIFY; IFT81: 1:1000, Cat No: 11744-1-AP, ProteinTech; β-actin: 1:2000, Cat No: 4967 S, Cell Signaling Antibodies against IFT20, IFT27, IFT57, IFT88 and IFT140 (1:2000) were from Dr. Pazour’s laboratory ( , , ); AKAP4: 1:4000, from Dr. George Gerton at University of Pennsylvania; ODF2: 1:800, Cat No: 12058-1-AP, ProteinTech; SPAG16L: 1:1000, generated by Z.Z.’s laboratory) at 4°C overnight.

Techniques: Expressing, Knock-Out, Western Blot

Journal: Discover Oncology

Article Title: Hsa_circ_0075451 promotes NSCLC proliferation, metastasis, and glycolysis through interaction with the RNA-binding protein RBM4

doi: 10.1007/s12672-025-03330-4

Figure Lengend Snippet: Interaction between hsa_circ_0075451 and RBM4. A GEPIA database (http://www.gepia.cancer‑pku.cn/index.html) analysis of the differential expression of RBM4 in LUAD/LUSC tumor tissues and normal tissues; B GEPIA database analysis of the correlation between RBM4 and GMDS expression in LUAD/LUSC tumor tissues, and calculation of the Pearson correlation coefficient; C RT-qPCR detection of RBM4 mRNA expression levels in NSCLC tissues and adjacent normal tissues ( N = 53); D Pearson correlation analysis of RBM4 mRNA and hsa_circ_0075451 expression levels in NSCLC tissues ( N = 53); E RT-qPCR analysis of RBM4 expression levels in the human normal lung epithelial cell line BEAS-2B and NSCLC cell lines PC9 and A549; F RNA pull-down assay using sense and antisense hsa_circ_0075451 probes, followed by WB detection to confirm the interaction between RBM4 and hsa_circ_0075451; G RIP assay using RBM4 antibody in cells, with IgG as a negative control, followed by RT-qPCR analysis to determine the enrichment of hsa_circ_0075451 in the RIP precipitates; H - I RT-qPCR and Western blot analysis of IGF2BP2 mRNA and protein expression levels after hsa_circ_0075451 downregulation in cells. All cellular experiments were performed with three independent biological replicates ( N = 3). * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: After blocking, they were incubated overnight at 4 °C with primary antibodies against RBM4 (1:1000, 11614-1-AP, Proteintech), Ki67 (1:1000, ab16667, Abcam, Cambridge, MA, USA), Bax (1:1000, ab32503, Abcam), MMP9 (1:1000, ab283575, Abcam), glucose transporter 1 (GLUT1) (1:10000, ab115730, Abcam), hexokinase 2 (HK2, 1:1000; ab227198, Abcam), and β-actin (1:1000; ab8226, Abcam), along with HRP-conjugated secondary antibodies (1:5000, Abcam) at room temperature for 1 h. The visualization of protein bands was achieved using enhanced chemiluminescence, and their quantification was done by densitometric analysis using ImageJ software.

Techniques: Quantitative Proteomics, Expressing, Quantitative RT-PCR, Pull Down Assay, Negative Control, Western Blot

Journal: Discover Oncology

Article Title: Hsa_circ_0075451 promotes NSCLC proliferation, metastasis, and glycolysis through interaction with the RNA-binding protein RBM4

doi: 10.1007/s12672-025-03330-4

Figure Lengend Snippet: Knockdown of RBM4 inhibits NSCLC cell proliferation, migration, and glycolysis. A Western blot detection of RBM4 expression levels in cells; B - C CCK-8 and EDU assays for assessing cell proliferation; D Annexin V-PI dual staining method for apoptosis detection; E Transwell assay for cell migration and invasion; F Western blot analysis of Ki67, Bax, and MMP9 protein expression levels in cells; G - H Assay kits measuring glucose uptake and lactate production in cells; I Western blot analysis of GLUT1 and HK2 protein expression levels in cells. All cellular experiments were performed with three independent biological replicates ( N = 3). * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: After blocking, they were incubated overnight at 4 °C with primary antibodies against RBM4 (1:1000, 11614-1-AP, Proteintech), Ki67 (1:1000, ab16667, Abcam, Cambridge, MA, USA), Bax (1:1000, ab32503, Abcam), MMP9 (1:1000, ab283575, Abcam), glucose transporter 1 (GLUT1) (1:10000, ab115730, Abcam), hexokinase 2 (HK2, 1:1000; ab227198, Abcam), and β-actin (1:1000; ab8226, Abcam), along with HRP-conjugated secondary antibodies (1:5000, Abcam) at room temperature for 1 h. The visualization of protein bands was achieved using enhanced chemiluminescence, and their quantification was done by densitometric analysis using ImageJ software.

Techniques: Knockdown, Migration, Western Blot, Expressing, CCK-8 Assay, Staining, Transwell Assay

Journal: Discover Oncology

Article Title: Hsa_circ_0075451 promotes NSCLC proliferation, metastasis, and glycolysis through interaction with the RNA-binding protein RBM4

doi: 10.1007/s12672-025-03330-4

Figure Lengend Snippet: Overexpression of RBM4 reverses the inhibitory effects of hsa_circ_0075451 knockdown on NSCLC cell proliferation, migration, and glycolysis. A RT-qPCR and Western blot analysis of RBM4 expression levels in cells; B - C CCK-8 and EDU assays for evaluating cell proliferation; D Annexin V-PI dual staining for apoptosis detection; E Transwell assay for assessing cell migration and invasion; F Western blot analysis of Ki67, Bax, and MMP9 protein expression levels in cells; G - H Assay kits for measuring glucose uptake and lactate production in cells; I Western blot analysis of GLUT1 and HK2 protein expression levels in cells. All cellular experiments were performed with three independent biological replicates ( N = 3). * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: After blocking, they were incubated overnight at 4 °C with primary antibodies against RBM4 (1:1000, 11614-1-AP, Proteintech), Ki67 (1:1000, ab16667, Abcam, Cambridge, MA, USA), Bax (1:1000, ab32503, Abcam), MMP9 (1:1000, ab283575, Abcam), glucose transporter 1 (GLUT1) (1:10000, ab115730, Abcam), hexokinase 2 (HK2, 1:1000; ab227198, Abcam), and β-actin (1:1000; ab8226, Abcam), along with HRP-conjugated secondary antibodies (1:5000, Abcam) at room temperature for 1 h. The visualization of protein bands was achieved using enhanced chemiluminescence, and their quantification was done by densitometric analysis using ImageJ software.

Techniques: Over Expression, Knockdown, Migration, Quantitative RT-PCR, Western Blot, Expressing, CCK-8 Assay, Staining, Transwell Assay