Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELMalpha) induces the vascular and hemodynamic changes of pulmonary hypertension.

doi: 10.1152/ajplung.90526.2008

Figure Lengend Snippet: Fig. 1. In vivo knockdown of hypoxia-in- duced mitogenic factor (HIMF) in the chronic hypoxia model of pulmonary hypertension. A: real-time PCR for green fluorescent protein (GFP) from RNA isolated from the lung, heart, liver, and spleen 72 h after intratracheal instillation of Ad-HIMF-shRNA. B: equally divided left lungs from uninfected rats or rats infected with Ad-Neg-shRNA 4 days earlier were homogenized and evaluated for GFP ex- pression. The homogenates were resolved by 4–20% SDS-PAGE and transferred to nitro- cellulose. The blots were probed with rabbit polyclonal anti-GFP antibodies and developed with ECL. To confirm equal protein loading and transfer, the blots were stripped and re- probed with monoclonal anti- -actin antibod- ies. IB, immunoblot; IB*, immunoblot after stripping. C: visualization of GFP in control rat lungs or lungs infected with Ad-Neg- shRNA. DIC, differential interference contrast. D: lung homogenates from animals exposed to 4 days of normoxia (20.8% O2) or hypoxia (10.0% O2) were evaluated for HIMF expression. The homogenates were resolved by 4–20% SDS- PAGE and transferred to nitrocellulose. The blots were probed with polyclonal anti-HIMF antibodies and developed by ECL. To confirm introduction of the viral vectors, the blot was stripped and reprobed with polyclonal anti-GFP antibodies. To confirm equal protein loading, the blots were stripped and reprobed with monoclo- nal anti- -actin antibodies. IB**, immunoblot after second stripping.

Article Snippet: Nonspecific protein binding was blocked by treatment of the slides with normal rabbit serum for 30 min. After the final blocking step, the sections were treated with polyclonal goat anti-mouse RELM (1: 200; R&D Systems, Minneapolis, MN) or antibody diluent alone for 120 min at RT.

Techniques: In Vivo, Knockdown, Real-time Polymerase Chain Reaction, Isolation, shRNA, Infection, SDS Page, Western Blot, Stripping Membranes, Control, Expressing

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELMalpha) induces the vascular and hemodynamic changes of pulmonary hypertension.

doi: 10.1152/ajplung.90526.2008

Figure Lengend Snippet: Fig. 5. Effectiveness of pulmonary HIMF gene transfer. A: 2 wk after rats were intratra- cheally instilled with AAV-HIMF, the lungs were homogenized, resolved by 4–20% SDS- PAGE, and transferred to nitrocellulose. The blots were probed with rabbit anti-HIMF anti- bodies and developed by ECL. To confirm equal loading and transfer, the blot was stripped and reprobed with anti- -actin monoclonal an- tibodies. B: laser densitometry was used to quantify HIMF levels in the lung samples. Data were normalized to -actin expression and ex- pressed as relative intensity (means SE). The number of lungs studied is indicated within each bar. *P 0.05 compared with the control lung. C–E: paraffin-embedded lung sections from AAV-null-treated (C) and AAV-HIMF- treated (D and E) mice were rehydrated and stained with goat anti-mouse HIMF polyclonal antibodies. Scale bar 50 m.

Article Snippet: Nonspecific protein binding was blocked by treatment of the slides with normal rabbit serum for 30 min. After the final blocking step, the sections were treated with polyclonal goat anti-mouse RELM (1: 200; R&D Systems, Minneapolis, MN) or antibody diluent alone for 120 min at RT.

Techniques: SDS Page, Expressing, Control, Staining